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Selective Potentiation of Angiotensin-Induced Constriction of Skeletal Muscle Resistance Arteries by Chronic Elevations in Dietary Salt Intake
Microvascular Research 57, 310 –319 (1999) Article ID mvre.1999.2147, available online at http://www.idealibrary.com on
Selective Potentiation of Angiotensin-Induced Constriction of Skeletal Muscle Resistance Arteries by Chronic Elevations in Dietary Salt Intake David S. Weber, Jefferson C. Frisbee, and Julian H. Lombard Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received August 19, 1998
Sprague–Dawley rats were fed either a high-salt (HS, 4.0% NaCl) or a low-salt (LS, 0.4% NaCl) diet for 3 days (short-term) or 4 – 8 weeks (chronic). Vasoconstrictor responses to angiotensin II and norepinephrine were determined in isolated skeletal muscle resistance arteries and in distal arterioles of the in situ cremaster muscle. Myogenic responses to increases in transmural pressure were also assessed in skeletal muscle resistance arteries of animals on high- or lowsalt diets. Chronic (but not short-term) HS diet selectively potentiated angiotensin II-induced constriction of skeletal muscle resistance arteries relative to vessels from LS controls. Myogenic responses and norepinephrine-induced constriction of resistance arteries were unaffected by either chronic or short-term HS diet. Constriction of cremasteric arterioles in response to angiotensin II and norepinephrine was unaffected by chronic or short-term elevations in dietary salt intake. These data suggest that chronic elevations in dietary salt intake lead to a selective increase in the constriction of skeletal muscle resistance arteries to angiotensin II that may allow these vessels to continue to regulate their tone in response to this peptide, despite the suppression of angiotensin II that occurs with high-salt diet. © 1999 Academic Press Key Words: sodium; microcirculation; vascular smooth muscle; vasoconstriction.
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INTRODUCTION
Previous studies have demonstrated that chronic (4 – 8 weeks) high-salt diet results in significant structural alterations in microvessels (Greene et al., 1990; Hansen-Smith et al., 1990) and impaired relaxation of both resistance arteries and cremasteric arterioles in response to vasodilator stimuli (Frisbee and Lombard, 1998; Liu et al., 1997; Weber et al., 1997). More recent studies in our laboratory have demonstrated that alterations in microvascular structure (Hansen-Smith et al., 1996) and impaired vasodilator responses of resistance arteries (Weber et al., 1997) and microvessels (Frisbee and Lombard, 1999) develop very rapidly, i.e., after only 3 days of elevation in dietary salt intake. It has also been suggested that dietary salt may affect the levels of basal tone in the microvasculature of the spinotrapezius muscle (Boegehold, 1993a). These alterations in vasodilator responses or in the level of resting tone either could be a precursor to the onset of salt-sensitive hypertension or could contribute to an impaired ability of individuals on a high-salt diet to respond to cardiovascular challenges, such as exercise, reduced blood flow, and arterial hypoxemia. An important question regarding the effect of dietary salt intake on the regulation of vascular tone is whether elevated dietary salt intake also affects the 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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response of resistance arteries and microvessels to vasoconstrictor stimuli. Enhanced vasoconstrictor responses following elevations in dietary salt intake could be another mechanism by which a high-salt diet could result in elevations in total peripheral resistance and ultimately contribute to the onset or maintenance of hypertension. However, very little is known regarding the effect of elevated salt intake on vascular responses to constrictor stimuli in normotensive animals. Therefore, the major goals of the current study were to determine whether an elevation in dietary salt intake per se affects the sensitivity of skeletal muscle resistance arteries and arterioles to the sympathetic neurotransmitter norepinephrine and the vasoactive peptide angiotensin II and whether elevated dietary salt intake affects the myogenic response of skeletal muscle resistance arteries to increases in transmural pressure.
MATERIALS AND METHODS Experimental Animals Male Sprague–Dawley rats (Harlan, Madison, WI) were used for all studies. Rats were fed either a highsalt (HS, 4.0% NaCl) or a low-salt (LS, 0.4% NaCl) diet (Dyets, Inc., Bethlehem, PA) with tap water to drink ad libitum. This feeding regimen was maintained for either 3 days (short-term) or 4 – 8 weeks (chronic) prior to the studies. The characteristics of the different experimental groups used in these studies are summarized in Table 1.
Isolated Vessel Studies General procedures. On the day of the experiment, the rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg; Abbot Laboratories, 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 was carefully removed (Fredricks et al., 1994; Liu et al., 1997).
TABLE 1 Experimental Groups for Vascular Reactivity Studies Vessel type
Experiment duration
Microvessels
Short-term Chronic
Resistance arteries
Short-term Chronic
N
Age (weeks)
Weight (g)
Diet
12 12 12 12
12–13 12–13 16–17 16–17
356 6 18 362 6 19 486 6 20 474 6 18
LS HS LS HS
9 10 11 11
9–10 9–10 13–14 13–14
278 6 8 270 6 7 391 6 10 382 6 12
LS HS LS HS
Note. LS represents low-salt rat chow (0.4% NaCl) and HS represents high-salt rat chow (4.0% NaCl).
Particular care was taken to handle the vessel by the connective tissue at the ends and to minimize stretching of the vessel during removal. The isolated artery was then placed in warmed physiological salt solution (PSS) bubbled with 21% O 2, 5% CO 2, and 74% N 2. The PSS used in these experiments had the following constituents (mM): NaCl, 119; KCl, 4.7; MgSO 4 , 1.17; CaCl 2, 1.6; NaH 2PO 4, 1.18; NaHCO 3, 24; EDTA, 0.026; and glucose, 5.5. After isolation, the vessel was placed in a heated (37°C) chamber that allowed the lumen of the artery to be perfused with PSS and the outside of the vessel to be superfused with PSS from separate reservoirs. The artery was cannulated at both ends with tapered glass micropipettes (100 –150-mm in diameter) and secured onto the inflow and outflow pipettes using 10-0 nylon suture (22 mm in diameter; Look Inc., Norwell, MA). Any side branches were tied off with a single strand teased from 6-0 silk suture (Ethicon Inc., Somerville, NJ). The inflow pipette was connected to a reservoir perfusion system that allowed the intraluminal pressure and luminal gas concentrations to be controlled. Vessel diameters were measured utilizing television microscopy and an on-screen video microscaler (Model IV-550; FOR.A Co., Tokyo, Japan). After mounting on the micropipettes, the artery was extended to its in situ length and equilibrated at 100 mm Hg to approximate the pressure encountered in vivo. The viability of the artery was assessed by verifying that the vessel constricted following administration of 1 mM norepinephrine to the vessel chamber.
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After the viability of the vessel was determined, the norepinephrine was washed out for 10 min and the artery was equilibrated for 30 min with continuous superfusion and perfusion with PSS. Any vessel that did not constrict in response to norepinephrine or that did not show active tone at rest was not used in the study. Active tone (percentage) at the control equilibration pressure (100 mm Hg) was calculated as (DD/ D max) 3 100, where DD is the diameter increase from the resting value in response to the Ca 21-free relaxing solution, and D max is the maximum diameter measured at the control equilibration pressure in the Ca 21-free relaxing solution. Active tone in these vessels averaged 19.6 6 3.0% in the short-term LS animals (n 5 17), 21.1 6 2.49% in the short-term HS animals (n 5 18), 14.4 6 1.1% in the chronic LS animals (n 5 17), and 15.5 6 1.9% in the chronic HS animals (n 5 17). Response of skeletal muscle resistance arteries to constrictor agonists. Following a minimum of 30 min equilibration in PSS at 100 mm Hg, the responses of the vessels to norepinephrine (10 211–10 26 M) and the human analog of angiotensin II (10 211–10 27 M) were determined by measuring vascular diameters before and after addition of the agonist to the tissue bath. The norepinephrine and angiotensin II used in these experiments were both purchased from Sigma Chemical Co. (St. Louis, MO). The diameter that was measured immediately before the drug was added to the bath served as the control value. To administer the drug, the flow of superfusion solution in the bath was briefly interrupted and the drug was added to the PSS to achieve the desired concentration in the vessel chamber. Vessel diameter was monitored constantly and the reported values are the maximum constriction in response to each respective concentration of the drug. After each application of the vasoconstrictor agonist, the arteries were allowed to recover to the control diameter before the next dose of drug was administered. Application of the agents was randomized to prevent the occurrence of ordering effects. Myogenic responses. After equilibration of the vessels in PSS at the control pressure of 100 mm Hg, the outflow was clamped and the perfusion reservoir was lowered to achieve an intralumenal pressure of 0 mm Hg. The reservoir was then raised to produce 20-mm Hg stepwise increases in intralumenal pressure. Fol-
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Weber, Frisbee, and Lombard
lowing a brief equilibration (3 min) at each pressure step, vessel diameters were measured using video microscopy across a pressure range of 0 to 160 mm Hg. Myogenic responses were assessed by determining the ability of the vessel to maintain its diameter in response to increases in intralumenal pressure.
Microcirculation Studies Methods and protocols. On the day of the experiment, individual rats were anesthetized with an injection (60 mg/kg, i.p.) of sodium pentobarbital, and the trachea was cannulated to insure a patent airway. The carotid artery and external jugular vein were cannulated for arterial pressure recording and intravenous infusion of supplemental anesthetic as necessary. After the initial surgery was completed, the cremaster muscle was prepared for television microscopy, as described previously (Lombard et al., 1989). After completion of the in situ cremaster muscle preparation, the tissue was continuously superfused with physiological salt solution, equilibrated with a gas mixture of 5% CO 2 and 95% N 2, and maintained at 34 –35°C as it flowed over the muscle. The ionic composition of the PSS was as follows (mM): NaCl, 119.0; KCl, 4.7; CaCl 2 , 1.6; NaH 2 PO 4 , 1.18; MgSO 4 , 1.17; NaHCO 3, 24.0; and disodium EDTA, 0.03. Arteriolar diameters were determined with a videomicrometer, accurate to 61 mm (Lombard et al., 1989). Arterioles selected for these studies were distal (third or fourth order) arterioles of the cremaster muscle that were situated in areas of the muscle that were away from the edges of the incision and that had clearly visible walls, brisk flow velocity, and active tone, as judged by the occurrence of a brisk dilation in response to topical application of 10 24 M adenosine. Response of the microvasculature to vasoconstrictor agonists. Arteriolar responses to the topical application of either norepinephrine (10 210–10 26 M) or angiotensin II (10 29–10 26 M) were determined by using video microscopy. Successive agonist challenges were applied only after the vessel had returned to its resting diameter following the application of the preceding agonist. Application of the agents was randomized to prevent the occurrence of ordering effects.
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groups used in these studies were 152 6 4 mm (n 5 17) for short-term LS, 146 6 3 mm (n 5 18) for short-term HS, 157 6 5 mm (n 5 17) for chronic LS, and 158 6 5 mm (n 5 17) for chronic HS. Mean control diameters (6 SE) of cremasteric arterioles in the various groups used in these studies were 18 6 1 mm (n 5 12) for short-term LS, 20 6 2 mm (n 5 12) for short-term HS, 22 6 1 mm (n 5 12) for chronic LS, and 26 6 2 mm (n 5 12) for chronic HS.
Response to Angiotensin II FIG. 1. Mean arterial blood pressure in rats maintained on shortterm (3 days) or chronic (4 – 6 weeks) low-salt or high-salt diet. Data are expressed as mean 6 SE for 12 rats in each of the short-term groups and 23 rats in each of the chronic groups. There were no significant differences in arterial blood pressure as a function of dietary salt intake in either of the groups.
Statistical Analyses All data are presented as mean 6 SE. Myogenic response data were expressed as the absolute diameter in microns, while the responses to vasoconstrictor stimuli were expressed as the percentage decrease in vessel diameter from the control values. Analysis of variance (ANOVA) was employed to determine differences across experimental conditions. Duncan’s and Scheffe’s post hoc tests were employed to identify significant differences between specific experimental groups. In all cases, a probability level of P , 0.05 was considered to be statistically significant.
RESULTS Mean Arterial Pressure and Vessel Diameter Figure 1 summarizes the mean arterial pressure data from the various groups of animals used in this study. Mean arterial pressure in animals on high-salt and low-salt diets was not significantly different in either the short-term or the chronic groups. Mean control diameters (6 SE) for the gracilis arteries in the various
Figure 2 summarizes the response of gracilis arteries to increasing concentrations of angiotensin II in rats maintained on chronic (Fig. 2A) and short-term (Fig. 2B) high-salt or low-salt diets. As the concentration of angiotensin II was increased, the constriction of resistance arteries from rats maintained on the chronic high-salt diet (Fig. 2A) was significantly greater than those of their low-salt counterparts. In contrast, shortterm elevation of dietary salt intake did not potentiate the vasoconstrictor response to angiotensin II in these vessels (Fig. 2B). The effect of chronic and short-term exposure to high-salt diet on the constriction of cremasteric arterioles in response to angiotensin II is summarized in Fig. 3. In contrast to the potentiation of angiotensin II-induced constriction of resistance arteries during chronic exposure to a high-salt diet (Fig. 2), neither chronic (Fig. 3A) nor short-term (Fig. 3B) exposure to a high-salt diet affected the response of cremasteric arterioles to angiotensin II.
Response to Norepinephrine The vasoconstrictor responses to norepinephrine in gracilis arteries and cremasteric arterioles of animals subjected to chronic and short-term elevations in dietary salt intake are summarized in Figs. 4 and 5, respectively. Neither chronic nor short-term exposure to a high-salt diet affected the response to norepinephrine in skeletal muscle resistance arteries or cremasteric arterioles.
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Weber, Frisbee, and Lombard
Myogenic Responses in Resistance Arteries Figure 6 presents the response of skeletal muscle resistance arteries to increases in transmural pressure in these experiments. The gracilis artery is a muscular resistance artery that exhibits a high level of resting tone and a pronounced myogenic response, as evidenced by its ability to maintain its diameter in re-
FIG. 2. (A) Constriction of gracilis arteries (n 5 11 in each group) in response to increasing concentrations of angiotensin II (10 211–10 27 M) in rats maintained on a chronic LS diet or a chronic HS diet. (B) Constriction of gracilis arteries in response to increasing concentrations of angiotensin II (10 211–10 28 M) in rats maintained on a shortterm LS diet (n 5 9) or a short-term HS diet (n 5 10). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. Asterisk denotes a significant difference (P , 0.05) from respective LS controls determined by a 2-way ANOVA with a Duncan’s test a posteriori.
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FIG. 3. (A) Constriction of cremasteric arterioles in response to increasing concentrations of angiotensin II (10 29–10 26 M) in rats maintained on a chronic LS diet (n 5 6) or a chronic HS diet (n 5 5). (B) Constriction of cremasteric arterioles in response to increasing concentrations of angiotensin II (10 29–10 26 M) in rats maintained on a short-term LS diet (n 5 8) or a short-term HS diet (n 5 7). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. There was no significant difference between rats on HS or LS diet in either group of experiments.
sponse to increasing levels of transmural pressure. Although the arteries used in these experiments had substantial amounts of active tone, maintenance of the animals on either a chronic high-salt diet (Fig. 6A) or a short-term high-salt diet (Fig. 6B) had no effect on the resting tone or the myogenic response of the vessel to increases in transmural pressure.
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ies (Liu et al., 1997; Weber et al., 1997) and skeletal muscle arterioles (Boegehold, 1993b; Frisbee and Lombard, 1998 1999) are impaired by elevations in dietary salt intake. Therefore, a natural question is whether the responses of these vessels to vasoconstrictor stimuli are also affected by increases in dietary salt intake. This is important because alterations in the response of resistance arteries and arterioles to different vaso-
FIG. 4. (A) Constriction of gracilis arteries (n 5 11 in each group) in response to increasing concentrations of norepinephrine (10 211– 10 26 M) in rats maintained on a chronic LS diet or a chronic HS diet. (B) Constriction of gracilis arteries in response to increasing concentrations of norepinephrine (10 211–10 26 M) in rats maintained on a short-term LS diet (n 5 9) or a short-term HS diet (n 5 10). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. There was no significant difference between rats on HS or LS diet in either group of experiments.
DISCUSSION Previous studies by our group (Greene et al., 1990; Hansen-Smith et al., 1990 1996; Frisbee and Lombard, 1998) and by other authors (Boegehold, 1993a) have demonstrated that a high-salt diet leads to significant alterations in the structure of skeletal muscle arterioles and that the vasodilator responses of resistance arter-
FIG. 5. (A) Constriction of cremasteric arterioles in response to increasing concentrations of norepinephrine (10 210–10 26 M) in rats maintained on a chronic LS diet (n 5 6) or a chronic HS diet (n 5 5). (B) Constriction of cremasteric arterioles in response to increasing concentrations of norepinephrine (10 210–10 26 M) in rats maintained on a short-term LS diet (n 5 8) or a short-term HS diet (n 5 7). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. There was no significant difference between rats on HS or LS diet in either group of experiments.
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FIG. 6. Comparison of the myogenic response to transmural pressure elevation in skeletal muscle resistance arteries of rats maintained on chronic and short-term high-salt and low-salt diets. Groups of rats were maintained on either a HS (n 5 22) or LS (n 5 19) diet for 4 – 8 weeks (A) or a short-term (3 days) HS (n 5 10) or LS (n 5 17) diet (B). Data are expressed as mean diameter (6 SE) at each transmural pressure. The graphs show internal diameters (mm) of rat gracilis arteries equilibrated in physiological salt solution (PSS) and relaxing solution of Ca 21-free PSS.
constrictor and vasodilator stimuli may suggest mechanisms for changes in vascular function that could serve as a precursor to the elevation in total peripheral resistance in salt-sensitive forms of hypertension.
Vasoconstrictor Responses to Angiotensin II In the present study, we demonstrated that the constriction of skeletal muscle resistance arteries in response to angiotensin II is enhanced with chronic (but not short-term) elevations in dietary salt intake (Fig. 2). These findings are consistent with previous reports in the literature that sodium restriction decreases vascular responsiveness to angiotensin II while sodium loading increases vascular angiotensin II sensitivity
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Weber, Frisbee, and Lombard
(Aguilera and Catt, 1981; Catt et al., 1984; Yoshida et al., 1987). Taken together, these findings clearly suggest that there is a correlation between dietary sodium levels and the constrictor responses to angiotensin II. One question arising from the present experiments is why the angiotensin II concentrations required to elicit vasoconstriction in the present study (and in many others) are so much higher than normal circulating levels of angiotensin II, which are in the picomolar range (Rieder et al., 1997). One possible explanation is that angiotensin II levels near the vascular angiotensin receptors may be higher than those in the plasma, possibly because of the presence of a local renin–angiotensin system in the vessel wall. Regardless of the reason(s) for the difference between circulating levels of angiotensin II and the angiotensin II concentrations required to cause constriction of isolated or in situ blood vessels, the presence of an enhanced constriction in response to angiotensin II in gracilis arteries of animals on a chronic high-salt diet suggests that dietary salt intake is a key factor determining the responsiveness of resistance arteries to the constrictor effects of this peptide. In these experiments, we demonstrated that an enhanced constriction in response to angiotensin II occurred only in the resistance arteries, since angiotensin-induced constriction of cremasteric arterioles was not potentiated by chronic exposure to a high-salt diet (Fig. 3). The enhanced responsiveness to the vasoconstrictor effects of angiotensin II in resistance arteries of animals on a high-salt diet was specific for this peptide, since chronic exposure to high-salt diet did not affect the response of the vessels to norepinephrine and transmural pressure elevation (see below). Taken together, these observations suggest that there is a selective potentiation of the vasoconstrictor response of skeletal muscle resistance arteries to angiotensin II during chronic elevations in dietary salt intake. The mechanism for the enhanced constriction of skeletal muscle resistance arteries in response to angiotensin II after a chronic elevation in dietary salt intake remains to be determined. Previous studies (Aguilera and Catt, 1981; Catt et al., 1984) suggested that salt-induced changes in vascular sensitivity to angiotensin II are apparently due to changes in the density of angiotensin receptors, rather than the affin-
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Angiotensin II Constriction and High Salt
ity of the receptors for the peptide. It is tempting to hypothesize that the reduction in the circulating levels of angiotensin II that occurs in animals on a high-salt diet results in a compensatory upregulation of angiotensin II receptors in an attempt to maintain the interaction of angiotensin II with the vasculature. The regulation of angiotensin receptor density by changes in dietary sodium varies from tissue to tissue, and some questions have arisen as to whether changes in larger vessels, such as the aorta, are representative of those occurring in smaller resistance vessels (Catt et al., 1984). Studies in the rat mesenteric artery suggest that changes in angiotensin II receptor density in the small resistance arteries may be similar to those in large arteries, such as aorta (Catt et al., 1984). However, the selective potentiation of the vasoconstrictor response to angiotensin II by high-salt diet in skeletal muscle resistance arteries (but not arterioles) in the present study suggests that salt-induced changes in the expression of angiotensin receptors may differ in these vessels. It is possible that the difference in the effect of high-salt diet on the vasoconstrictor response to angiotensin II in the resistance arteries and arterioles in the present study is due to changes in the relative proportions of the different angiotensin II receptor subtypes during changes in dietary salt intake. Previous studies have suggested that the AT 1 and AT 2 angiotensin receptor subtypes have opposing effects on blood pressure (Schuer and Perone, 1993; Munzenmaier and Greene, 1996; Unger et al., 1996) and vascular growth (Munzenmaier and Greene, 1996; Stoll et al., 1995a,b; Unger et al., 1996), i.e., the AT 1 receptor subtype mediates vasoconstriction and stimulates vascular growth, while the AT 2 receptor subtype mediates vasodilation and is growth inhibitory. The studies of Munzenmaier and Greene (1996) indicate that both the AT 1 and the AT 2 angiotensin receptor subtypes are present in cremasteric arterioles. Therefore, the difference in the effect of high-salt diet on angiotensininduced constriction of skeletal muscle resistance arteries and cremasteric arterioles in the present study could be due, at least in part, to differences in the distribution of the AT 1 and AT 2 receptors and/or a differential regulation of the AT 1 and AT 2 receptor subtypes in these vessels. In the case of the cremasteric
arterioles, this could result in a shift in the balance of the vasoconstrictor (AT 1) and vasodilator (AT 2) receptors, which would prevent the enhanced constriction of the arterioles in response to angiotensin II. However, the effect of changes in dietary salt intake on the density of different angiotensin II receptor subtypes in the microcirculation and the ultimate effect of changes in these receptor subtypes on microvascular regulation remain to be completely determined. The latter questions appear to be an important area for future investigation. Another important finding of the present study was that short-term elevations of dietary salt intake had no effect on the response of either resistance arteries (Fig. 2B) or cremasteric arterioles (Fig. 3B) to angiotensin II. These findings contrast with the rapid blunting of the vascular relaxation in response to a variety of vasodilator stimuli that was demonstrated in resistance arteries and cremasteric arterioles after only 3 days on a high-salt diet (Weber et al., 1997; Frisbee and Lombard, 1998 1999). Thus, the potentiation of angiotensin IIinduced vasoconstriction of resistance arteries by elevated dietary salt intake takes considerably longer to develop than the impaired dilator responses of small arteries that were demonstrated in previous studies.
Responses to Norepinephrine In contrast to the potentiation of angiotensin IIinduced constriction of skeletal muscle resistance arteries by chronic elevations in dietary salt intake, neither chronic nor short-term elevations in dietary salt intake affected norepinephrine-induced constriction of the resistance arteries (Fig. 4) or the cremasteric arterioles (Fig. 5) in the present study. These findings are consistent with the results of a previous study by Nilsson et al. (1985), who reported no change in the norepinephrine sensitivity of the isolated perfused hindquarters preparation of WKY and SHR rats maintained on low-, normal, and high-salt diets for 8 –10 weeks. In another study, Yoshida et al. (1987) reported no difference in norepinephrine-induced constriction in aortic and renal artery strips of rabbits maintained on low-, normal, and high-sodium diets. In contrast, Soltis et al. (1993) reported that the vasoconstrictor response to norepinephrine was enhanced in thoracic
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aortic rings of rats that were maintained on a high-salt diet for 4 weeks after weaning and then returned to normal salt levels for 10 weeks. However, the latter study was confounded by an increase in systolic blood pressure in the high-salt diet rats, suggesting that the change in norepinephrine sensitivity in that study resulted from the increase in arterial blood pressure, rather than the elevation in dietary salt intake.
Myogenic Response In the present study, we also tested the effect of elevated dietary salt intake on the myogenic response of skeletal muscle resistance arteries to transmural pressure elevation. The myogenic response is an intrinsic, non-agonist-dependent contractile response that occurs in response to elevated pressure in many blood vessels. Myogenic responses allow blood vessels to increase their level of active tone in order to maintain a constant diameter or to constrict in response to increases in intralumenal pressure. This is an important mechanism for the local regulation of blood flow and downstream perfusion pressure. In the current studies, neither chronic nor short-term exposure to a high-salt diet affected the myogenic activation of the vessels. This finding is in contrast to previous studies by Takenaka et al. (1992), who reported an impaired myogenic response of renal afferent arterioles in the isolated hydronephrotic kidney preparation of Dahl R rats on a high-salt diet relative to controls on a low-salt diet. This may reflect differences in the strains of rats, the experimental preparation, or the function of these specific vessel types. For example, renal afferent arterioles are much smaller than the skeletal muscle resistance arteries used in the present study, and hence, they may be more sensitive to changes in intralumenal pressure or other mediators of local blood flow regulation.
SUMMARY AND CONCLUSIONS The current experiments have demonstrated that chronic, but not short-term, elevations in dietary salt intake lead to a selective potentiation of the response
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Weber, Frisbee, and Lombard
of skeletal muscle resistance arteries to angiotensin II. The increased sensitivity of the resistance arteries to angiotensin II may allow these vessels to adjust their tone in response to changes in the circulating levels of this peptide, even when plasma angiotensin II levels are reduced in response to the high-salt diet. This may be important in the regulation of resistance and blood flow within the vasculature. The lack of a change in the sensitivity of cremasteric arterioles to angiotensin II may be a protective mechanism of the microvasculature to maintain tissue blood flow and nutrient delivery despite the reductions in microvessel density that occur due to microvascular rarefaction associated with the high-salt diet (Greene et al., 1990; HansenSmith et al., 1996). The lack of a change in the response of the resistance arteries to norepinephrine and transmural pressure elevation and the lack of an enhanced response of the cremasteric arterioles to norepinephrine suggest that alterations in vasoconstrictor responses during chronic elevations in dietary salt intake are not generalized, but are specific to vasoconstrictor stimuli that are sensitive to changes in dietary salt levels.
ACKNOWLEDGMENTS This study was supported by NIH Grants HL 29587, HL 37374, and HL 52211. The authors thank Kathy Valent, Jane Schimke, and Barbara Vasko for their fine secretarial assistance.
REFERENCES Aguilera, G., and Catt, K. (1981). Regulation of vascular angiotensin II receptors in the rat during altered sodium intake. Circ. Res. 49, 751–758. Boegehold, M. A. (1993a). Microvascular changes associated with high salt intake and hypertension in Dahl rats. Int. J. Microcirc. Clin. Exp. 12, 143–156. Boegehold, M. A. (1993b). Effect of dietary salt on arteriolar nitric oxide in striated muscle of normotensive rats. Am. J. Physiol. 264, H1810 –H1816. Catt, K. J., Mendelsohn, F. A. C., Millan, M. A., and Aguilera, G. (1984). The role of angiotensin II receptors in vascular regulation. J. Cardiovasc. Pharmacol. 6, S575–S586.
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Fredricks, K. T., Liu, Y., and Lombard, J. H. (1994). Response of extraparenchymal resistance arteries of rat skeletal muscle to reduced PO 2. Am. J. Physiol. 267, H706 –H715. Frisbee, J. C., and Lombard, J. H. (1998). Chronic elevations in salt intake and reduced renal mass hypertension compromise mechanisms of arteriolar dilation. Microvasc. Res. 56, 218 –227. Frisbee, J. C., and Lombard, J. H. (1999). Acute elevations in salt intake and reduced renal mass hypertension compromise mechanisms of arteriolar dilation. Microvasc. Res., in press. Greene, A. S., Lombard, J. H., Cowley, A. W., Jr., and Hansen-Smith, F. M. (1990). Microvessel changes in hypertension measured by Griffonia simplicifolia I lectin. Hypertension 15, 779 –783. Hansen-Smith, F. M., Greene, A. S., Cowley, A. W., Jr., and Lombard, J. H. (1990). Structural changes during microvascular rarefaction in chronic hypertension. Hypertension 15, 922–928. Hansen-Smith, F. M., Morris, L. W., Greene, A. S., and Lombard, J. H. (1996). Rapid microvessel rarefaction with elevated salt intake and reduced renal mass hypertension in rats. Circ. Res. 79, 324 –330. Liu, Y., Fredricks, K. T., Roman, R. J., and Lombard, J. H. (1997). Response of resistance arteries to reduced PO 2 and vasodilators during hypertension and elevated salt intake. Am. J. Physiol. 273, H869 –H877. Lombard, J. H., Hinojosa-Laborde, C., and Cowley, A. W. (1989). Hemodynamics and microcirculatory alterations in reduced renal mass hypertension. Hypertension 13, 128 –138. Meininger, G. A., and Davis, M. J. (1992). Cellular mechanisms involved in the vascular myogenic response. Am. J. Physiol. 263, H647–H659. Munzenmaier, D. H., and Greene, A. S. (1996). Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension 27 (Part 2), 760 –765. Nilsson, H., Ely, D., Friberg, P., Karlstrom, G., and Folkow, B. (1985). Effects of high and low sodium diets on the resistance vessels and
319 their adrenergic vasoconstrictor fibre control in normotensive (WKY) and hypertensive (SHR) rats. Acta Physiol. Scand. 125, 323–334. Rieder, M. J., Roman, R. J., and Greene, A. S. (1997). Reversal of microvascular rarefaction and reduced renal mass hypertension. Hypertension 30 (Part 1), 120 –127. Scheuer, D. A., and Perrone, M. H. (1993). Angiotensin type 2 receptors mediate depressor phase of biphasic pressure response to angiotensin. Am. J. Physiol. 264, R917–R923. Soltis, E. E., Bartrug, B. L., and Newman, P. S. (1993). Chronic hypertension and vascular alterations following short-term high dietary salt intake in postweaning rats. Dev. Pharmacol. Ther. 20, 107–110. Stoll, M., Meffert, S., Stroth, U., and Unger, T. (1995a). Growth or antigrowth: Angiotensin and the endothelium. J. Hypertens. 13, 1529 –1534. Stoll, M., Steckelings, U., Paul, M., Bottari, S. P., Metzger, R., and Unger, T. (1995b). The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J. Clin. Invest. 95, 651– 657. Takenaka, T., Forster, H., De Micheli, A., and Epstein, M. (1992). Impaired myogenic responsiveness of renal microvessels in dahl salt-sensitive rats. Circ. Res. 71, 471– 480. Unger, T., Chung, O., Csikos, T., Culman, J., Gallinat, S., Gohlke, P., Hohle, S., Meffert, S., Stoll, M., Stroth, U., and Zhu, Y. (1996). Angiotensin receptors. J. Hypertens. 14(Suppl. 5), S95–S103. Weber, D. S., Greene, A. S., and Lombard, J. H. (1997). Effect of short term and chronic high salt diet on vasodilator response of skeletal muscle resistance arteries. FASEB J. 11, A43. [Abstract] Yoshida, M., Ueda, S., Machida, J., and Ikegami, K. (1987). The change of vascular reactivity to angiotensin II and norepinephrine in the two-kidney, one-clip renovascular hypertensive rabbit. J. Urol. 137, 1048 –1052.
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Selective Potentiation of Angiotensin-Induced Constriction of Skeletal Muscle Resistance Arteries by Chronic Elevations in Dietary Salt Intake David S. Weber, Jefferson C. Frisbee, and Julian H. Lombard Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received August 19, 1998
Sprague–Dawley rats were fed either a high-salt (HS, 4.0% NaCl) or a low-salt (LS, 0.4% NaCl) diet for 3 days (short-term) or 4 – 8 weeks (chronic). Vasoconstrictor responses to angiotensin II and norepinephrine were determined in isolated skeletal muscle resistance arteries and in distal arterioles of the in situ cremaster muscle. Myogenic responses to increases in transmural pressure were also assessed in skeletal muscle resistance arteries of animals on high- or lowsalt diets. Chronic (but not short-term) HS diet selectively potentiated angiotensin II-induced constriction of skeletal muscle resistance arteries relative to vessels from LS controls. Myogenic responses and norepinephrine-induced constriction of resistance arteries were unaffected by either chronic or short-term HS diet. Constriction of cremasteric arterioles in response to angiotensin II and norepinephrine was unaffected by chronic or short-term elevations in dietary salt intake. These data suggest that chronic elevations in dietary salt intake lead to a selective increase in the constriction of skeletal muscle resistance arteries to angiotensin II that may allow these vessels to continue to regulate their tone in response to this peptide, despite the suppression of angiotensin II that occurs with high-salt diet. © 1999 Academic Press Key Words: sodium; microcirculation; vascular smooth muscle; vasoconstriction.
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INTRODUCTION
Previous studies have demonstrated that chronic (4 – 8 weeks) high-salt diet results in significant structural alterations in microvessels (Greene et al., 1990; Hansen-Smith et al., 1990) and impaired relaxation of both resistance arteries and cremasteric arterioles in response to vasodilator stimuli (Frisbee and Lombard, 1998; Liu et al., 1997; Weber et al., 1997). More recent studies in our laboratory have demonstrated that alterations in microvascular structure (Hansen-Smith et al., 1996) and impaired vasodilator responses of resistance arteries (Weber et al., 1997) and microvessels (Frisbee and Lombard, 1999) develop very rapidly, i.e., after only 3 days of elevation in dietary salt intake. It has also been suggested that dietary salt may affect the levels of basal tone in the microvasculature of the spinotrapezius muscle (Boegehold, 1993a). These alterations in vasodilator responses or in the level of resting tone either could be a precursor to the onset of salt-sensitive hypertension or could contribute to an impaired ability of individuals on a high-salt diet to respond to cardiovascular challenges, such as exercise, reduced blood flow, and arterial hypoxemia. An important question regarding the effect of dietary salt intake on the regulation of vascular tone is whether elevated dietary salt intake also affects the 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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response of resistance arteries and microvessels to vasoconstrictor stimuli. Enhanced vasoconstrictor responses following elevations in dietary salt intake could be another mechanism by which a high-salt diet could result in elevations in total peripheral resistance and ultimately contribute to the onset or maintenance of hypertension. However, very little is known regarding the effect of elevated salt intake on vascular responses to constrictor stimuli in normotensive animals. Therefore, the major goals of the current study were to determine whether an elevation in dietary salt intake per se affects the sensitivity of skeletal muscle resistance arteries and arterioles to the sympathetic neurotransmitter norepinephrine and the vasoactive peptide angiotensin II and whether elevated dietary salt intake affects the myogenic response of skeletal muscle resistance arteries to increases in transmural pressure.
MATERIALS AND METHODS Experimental Animals Male Sprague–Dawley rats (Harlan, Madison, WI) were used for all studies. Rats were fed either a highsalt (HS, 4.0% NaCl) or a low-salt (LS, 0.4% NaCl) diet (Dyets, Inc., Bethlehem, PA) with tap water to drink ad libitum. This feeding regimen was maintained for either 3 days (short-term) or 4 – 8 weeks (chronic) prior to the studies. The characteristics of the different experimental groups used in these studies are summarized in Table 1.
Isolated Vessel Studies General procedures. On the day of the experiment, the rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg; Abbot Laboratories, 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 was carefully removed (Fredricks et al., 1994; Liu et al., 1997).
TABLE 1 Experimental Groups for Vascular Reactivity Studies Vessel type
Experiment duration
Microvessels
Short-term Chronic
Resistance arteries
Short-term Chronic
N
Age (weeks)
Weight (g)
Diet
12 12 12 12
12–13 12–13 16–17 16–17
356 6 18 362 6 19 486 6 20 474 6 18
LS HS LS HS
9 10 11 11
9–10 9–10 13–14 13–14
278 6 8 270 6 7 391 6 10 382 6 12
LS HS LS HS
Note. LS represents low-salt rat chow (0.4% NaCl) and HS represents high-salt rat chow (4.0% NaCl).
Particular care was taken to handle the vessel by the connective tissue at the ends and to minimize stretching of the vessel during removal. The isolated artery was then placed in warmed physiological salt solution (PSS) bubbled with 21% O 2, 5% CO 2, and 74% N 2. The PSS used in these experiments had the following constituents (mM): NaCl, 119; KCl, 4.7; MgSO 4 , 1.17; CaCl 2, 1.6; NaH 2PO 4, 1.18; NaHCO 3, 24; EDTA, 0.026; and glucose, 5.5. After isolation, the vessel was placed in a heated (37°C) chamber that allowed the lumen of the artery to be perfused with PSS and the outside of the vessel to be superfused with PSS from separate reservoirs. The artery was cannulated at both ends with tapered glass micropipettes (100 –150-mm in diameter) and secured onto the inflow and outflow pipettes using 10-0 nylon suture (22 mm in diameter; Look Inc., Norwell, MA). Any side branches were tied off with a single strand teased from 6-0 silk suture (Ethicon Inc., Somerville, NJ). The inflow pipette was connected to a reservoir perfusion system that allowed the intraluminal pressure and luminal gas concentrations to be controlled. Vessel diameters were measured utilizing television microscopy and an on-screen video microscaler (Model IV-550; FOR.A Co., Tokyo, Japan). After mounting on the micropipettes, the artery was extended to its in situ length and equilibrated at 100 mm Hg to approximate the pressure encountered in vivo. The viability of the artery was assessed by verifying that the vessel constricted following administration of 1 mM norepinephrine to the vessel chamber.
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After the viability of the vessel was determined, the norepinephrine was washed out for 10 min and the artery was equilibrated for 30 min with continuous superfusion and perfusion with PSS. Any vessel that did not constrict in response to norepinephrine or that did not show active tone at rest was not used in the study. Active tone (percentage) at the control equilibration pressure (100 mm Hg) was calculated as (DD/ D max) 3 100, where DD is the diameter increase from the resting value in response to the Ca 21-free relaxing solution, and D max is the maximum diameter measured at the control equilibration pressure in the Ca 21-free relaxing solution. Active tone in these vessels averaged 19.6 6 3.0% in the short-term LS animals (n 5 17), 21.1 6 2.49% in the short-term HS animals (n 5 18), 14.4 6 1.1% in the chronic LS animals (n 5 17), and 15.5 6 1.9% in the chronic HS animals (n 5 17). Response of skeletal muscle resistance arteries to constrictor agonists. Following a minimum of 30 min equilibration in PSS at 100 mm Hg, the responses of the vessels to norepinephrine (10 211–10 26 M) and the human analog of angiotensin II (10 211–10 27 M) were determined by measuring vascular diameters before and after addition of the agonist to the tissue bath. The norepinephrine and angiotensin II used in these experiments were both purchased from Sigma Chemical Co. (St. Louis, MO). The diameter that was measured immediately before the drug was added to the bath served as the control value. To administer the drug, the flow of superfusion solution in the bath was briefly interrupted and the drug was added to the PSS to achieve the desired concentration in the vessel chamber. Vessel diameter was monitored constantly and the reported values are the maximum constriction in response to each respective concentration of the drug. After each application of the vasoconstrictor agonist, the arteries were allowed to recover to the control diameter before the next dose of drug was administered. Application of the agents was randomized to prevent the occurrence of ordering effects. Myogenic responses. After equilibration of the vessels in PSS at the control pressure of 100 mm Hg, the outflow was clamped and the perfusion reservoir was lowered to achieve an intralumenal pressure of 0 mm Hg. The reservoir was then raised to produce 20-mm Hg stepwise increases in intralumenal pressure. Fol-
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Weber, Frisbee, and Lombard
lowing a brief equilibration (3 min) at each pressure step, vessel diameters were measured using video microscopy across a pressure range of 0 to 160 mm Hg. Myogenic responses were assessed by determining the ability of the vessel to maintain its diameter in response to increases in intralumenal pressure.
Microcirculation Studies Methods and protocols. On the day of the experiment, individual rats were anesthetized with an injection (60 mg/kg, i.p.) of sodium pentobarbital, and the trachea was cannulated to insure a patent airway. The carotid artery and external jugular vein were cannulated for arterial pressure recording and intravenous infusion of supplemental anesthetic as necessary. After the initial surgery was completed, the cremaster muscle was prepared for television microscopy, as described previously (Lombard et al., 1989). After completion of the in situ cremaster muscle preparation, the tissue was continuously superfused with physiological salt solution, equilibrated with a gas mixture of 5% CO 2 and 95% N 2, and maintained at 34 –35°C as it flowed over the muscle. The ionic composition of the PSS was as follows (mM): NaCl, 119.0; KCl, 4.7; CaCl 2 , 1.6; NaH 2 PO 4 , 1.18; MgSO 4 , 1.17; NaHCO 3, 24.0; and disodium EDTA, 0.03. Arteriolar diameters were determined with a videomicrometer, accurate to 61 mm (Lombard et al., 1989). Arterioles selected for these studies were distal (third or fourth order) arterioles of the cremaster muscle that were situated in areas of the muscle that were away from the edges of the incision and that had clearly visible walls, brisk flow velocity, and active tone, as judged by the occurrence of a brisk dilation in response to topical application of 10 24 M adenosine. Response of the microvasculature to vasoconstrictor agonists. Arteriolar responses to the topical application of either norepinephrine (10 210–10 26 M) or angiotensin II (10 29–10 26 M) were determined by using video microscopy. Successive agonist challenges were applied only after the vessel had returned to its resting diameter following the application of the preceding agonist. Application of the agents was randomized to prevent the occurrence of ordering effects.
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groups used in these studies were 152 6 4 mm (n 5 17) for short-term LS, 146 6 3 mm (n 5 18) for short-term HS, 157 6 5 mm (n 5 17) for chronic LS, and 158 6 5 mm (n 5 17) for chronic HS. Mean control diameters (6 SE) of cremasteric arterioles in the various groups used in these studies were 18 6 1 mm (n 5 12) for short-term LS, 20 6 2 mm (n 5 12) for short-term HS, 22 6 1 mm (n 5 12) for chronic LS, and 26 6 2 mm (n 5 12) for chronic HS.
Response to Angiotensin II FIG. 1. Mean arterial blood pressure in rats maintained on shortterm (3 days) or chronic (4 – 6 weeks) low-salt or high-salt diet. Data are expressed as mean 6 SE for 12 rats in each of the short-term groups and 23 rats in each of the chronic groups. There were no significant differences in arterial blood pressure as a function of dietary salt intake in either of the groups.
Statistical Analyses All data are presented as mean 6 SE. Myogenic response data were expressed as the absolute diameter in microns, while the responses to vasoconstrictor stimuli were expressed as the percentage decrease in vessel diameter from the control values. Analysis of variance (ANOVA) was employed to determine differences across experimental conditions. Duncan’s and Scheffe’s post hoc tests were employed to identify significant differences between specific experimental groups. In all cases, a probability level of P , 0.05 was considered to be statistically significant.
RESULTS Mean Arterial Pressure and Vessel Diameter Figure 1 summarizes the mean arterial pressure data from the various groups of animals used in this study. Mean arterial pressure in animals on high-salt and low-salt diets was not significantly different in either the short-term or the chronic groups. Mean control diameters (6 SE) for the gracilis arteries in the various
Figure 2 summarizes the response of gracilis arteries to increasing concentrations of angiotensin II in rats maintained on chronic (Fig. 2A) and short-term (Fig. 2B) high-salt or low-salt diets. As the concentration of angiotensin II was increased, the constriction of resistance arteries from rats maintained on the chronic high-salt diet (Fig. 2A) was significantly greater than those of their low-salt counterparts. In contrast, shortterm elevation of dietary salt intake did not potentiate the vasoconstrictor response to angiotensin II in these vessels (Fig. 2B). The effect of chronic and short-term exposure to high-salt diet on the constriction of cremasteric arterioles in response to angiotensin II is summarized in Fig. 3. In contrast to the potentiation of angiotensin II-induced constriction of resistance arteries during chronic exposure to a high-salt diet (Fig. 2), neither chronic (Fig. 3A) nor short-term (Fig. 3B) exposure to a high-salt diet affected the response of cremasteric arterioles to angiotensin II.
Response to Norepinephrine The vasoconstrictor responses to norepinephrine in gracilis arteries and cremasteric arterioles of animals subjected to chronic and short-term elevations in dietary salt intake are summarized in Figs. 4 and 5, respectively. Neither chronic nor short-term exposure to a high-salt diet affected the response to norepinephrine in skeletal muscle resistance arteries or cremasteric arterioles.
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Weber, Frisbee, and Lombard
Myogenic Responses in Resistance Arteries Figure 6 presents the response of skeletal muscle resistance arteries to increases in transmural pressure in these experiments. The gracilis artery is a muscular resistance artery that exhibits a high level of resting tone and a pronounced myogenic response, as evidenced by its ability to maintain its diameter in re-
FIG. 2. (A) Constriction of gracilis arteries (n 5 11 in each group) in response to increasing concentrations of angiotensin II (10 211–10 27 M) in rats maintained on a chronic LS diet or a chronic HS diet. (B) Constriction of gracilis arteries in response to increasing concentrations of angiotensin II (10 211–10 28 M) in rats maintained on a shortterm LS diet (n 5 9) or a short-term HS diet (n 5 10). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. Asterisk denotes a significant difference (P , 0.05) from respective LS controls determined by a 2-way ANOVA with a Duncan’s test a posteriori.
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FIG. 3. (A) Constriction of cremasteric arterioles in response to increasing concentrations of angiotensin II (10 29–10 26 M) in rats maintained on a chronic LS diet (n 5 6) or a chronic HS diet (n 5 5). (B) Constriction of cremasteric arterioles in response to increasing concentrations of angiotensin II (10 29–10 26 M) in rats maintained on a short-term LS diet (n 5 8) or a short-term HS diet (n 5 7). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. There was no significant difference between rats on HS or LS diet in either group of experiments.
sponse to increasing levels of transmural pressure. Although the arteries used in these experiments had substantial amounts of active tone, maintenance of the animals on either a chronic high-salt diet (Fig. 6A) or a short-term high-salt diet (Fig. 6B) had no effect on the resting tone or the myogenic response of the vessel to increases in transmural pressure.
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ies (Liu et al., 1997; Weber et al., 1997) and skeletal muscle arterioles (Boegehold, 1993b; Frisbee and Lombard, 1998 1999) are impaired by elevations in dietary salt intake. Therefore, a natural question is whether the responses of these vessels to vasoconstrictor stimuli are also affected by increases in dietary salt intake. This is important because alterations in the response of resistance arteries and arterioles to different vaso-
FIG. 4. (A) Constriction of gracilis arteries (n 5 11 in each group) in response to increasing concentrations of norepinephrine (10 211– 10 26 M) in rats maintained on a chronic LS diet or a chronic HS diet. (B) Constriction of gracilis arteries in response to increasing concentrations of norepinephrine (10 211–10 26 M) in rats maintained on a short-term LS diet (n 5 9) or a short-term HS diet (n 5 10). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. There was no significant difference between rats on HS or LS diet in either group of experiments.
DISCUSSION Previous studies by our group (Greene et al., 1990; Hansen-Smith et al., 1990 1996; Frisbee and Lombard, 1998) and by other authors (Boegehold, 1993a) have demonstrated that a high-salt diet leads to significant alterations in the structure of skeletal muscle arterioles and that the vasodilator responses of resistance arter-
FIG. 5. (A) Constriction of cremasteric arterioles in response to increasing concentrations of norepinephrine (10 210–10 26 M) in rats maintained on a chronic LS diet (n 5 6) or a chronic HS diet (n 5 5). (B) Constriction of cremasteric arterioles in response to increasing concentrations of norepinephrine (10 210–10 26 M) in rats maintained on a short-term LS diet (n 5 8) or a short-term HS diet (n 5 7). All data are expressed as mean percentage decrease (6 SE) from control diameter measured during equilibration of the arteries in PSS. There was no significant difference between rats on HS or LS diet in either group of experiments.
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FIG. 6. Comparison of the myogenic response to transmural pressure elevation in skeletal muscle resistance arteries of rats maintained on chronic and short-term high-salt and low-salt diets. Groups of rats were maintained on either a HS (n 5 22) or LS (n 5 19) diet for 4 – 8 weeks (A) or a short-term (3 days) HS (n 5 10) or LS (n 5 17) diet (B). Data are expressed as mean diameter (6 SE) at each transmural pressure. The graphs show internal diameters (mm) of rat gracilis arteries equilibrated in physiological salt solution (PSS) and relaxing solution of Ca 21-free PSS.
constrictor and vasodilator stimuli may suggest mechanisms for changes in vascular function that could serve as a precursor to the elevation in total peripheral resistance in salt-sensitive forms of hypertension.
Vasoconstrictor Responses to Angiotensin II In the present study, we demonstrated that the constriction of skeletal muscle resistance arteries in response to angiotensin II is enhanced with chronic (but not short-term) elevations in dietary salt intake (Fig. 2). These findings are consistent with previous reports in the literature that sodium restriction decreases vascular responsiveness to angiotensin II while sodium loading increases vascular angiotensin II sensitivity
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Weber, Frisbee, and Lombard
(Aguilera and Catt, 1981; Catt et al., 1984; Yoshida et al., 1987). Taken together, these findings clearly suggest that there is a correlation between dietary sodium levels and the constrictor responses to angiotensin II. One question arising from the present experiments is why the angiotensin II concentrations required to elicit vasoconstriction in the present study (and in many others) are so much higher than normal circulating levels of angiotensin II, which are in the picomolar range (Rieder et al., 1997). One possible explanation is that angiotensin II levels near the vascular angiotensin receptors may be higher than those in the plasma, possibly because of the presence of a local renin–angiotensin system in the vessel wall. Regardless of the reason(s) for the difference between circulating levels of angiotensin II and the angiotensin II concentrations required to cause constriction of isolated or in situ blood vessels, the presence of an enhanced constriction in response to angiotensin II in gracilis arteries of animals on a chronic high-salt diet suggests that dietary salt intake is a key factor determining the responsiveness of resistance arteries to the constrictor effects of this peptide. In these experiments, we demonstrated that an enhanced constriction in response to angiotensin II occurred only in the resistance arteries, since angiotensin-induced constriction of cremasteric arterioles was not potentiated by chronic exposure to a high-salt diet (Fig. 3). The enhanced responsiveness to the vasoconstrictor effects of angiotensin II in resistance arteries of animals on a high-salt diet was specific for this peptide, since chronic exposure to high-salt diet did not affect the response of the vessels to norepinephrine and transmural pressure elevation (see below). Taken together, these observations suggest that there is a selective potentiation of the vasoconstrictor response of skeletal muscle resistance arteries to angiotensin II during chronic elevations in dietary salt intake. The mechanism for the enhanced constriction of skeletal muscle resistance arteries in response to angiotensin II after a chronic elevation in dietary salt intake remains to be determined. Previous studies (Aguilera and Catt, 1981; Catt et al., 1984) suggested that salt-induced changes in vascular sensitivity to angiotensin II are apparently due to changes in the density of angiotensin receptors, rather than the affin-
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ity of the receptors for the peptide. It is tempting to hypothesize that the reduction in the circulating levels of angiotensin II that occurs in animals on a high-salt diet results in a compensatory upregulation of angiotensin II receptors in an attempt to maintain the interaction of angiotensin II with the vasculature. The regulation of angiotensin receptor density by changes in dietary sodium varies from tissue to tissue, and some questions have arisen as to whether changes in larger vessels, such as the aorta, are representative of those occurring in smaller resistance vessels (Catt et al., 1984). Studies in the rat mesenteric artery suggest that changes in angiotensin II receptor density in the small resistance arteries may be similar to those in large arteries, such as aorta (Catt et al., 1984). However, the selective potentiation of the vasoconstrictor response to angiotensin II by high-salt diet in skeletal muscle resistance arteries (but not arterioles) in the present study suggests that salt-induced changes in the expression of angiotensin receptors may differ in these vessels. It is possible that the difference in the effect of high-salt diet on the vasoconstrictor response to angiotensin II in the resistance arteries and arterioles in the present study is due to changes in the relative proportions of the different angiotensin II receptor subtypes during changes in dietary salt intake. Previous studies have suggested that the AT 1 and AT 2 angiotensin receptor subtypes have opposing effects on blood pressure (Schuer and Perone, 1993; Munzenmaier and Greene, 1996; Unger et al., 1996) and vascular growth (Munzenmaier and Greene, 1996; Stoll et al., 1995a,b; Unger et al., 1996), i.e., the AT 1 receptor subtype mediates vasoconstriction and stimulates vascular growth, while the AT 2 receptor subtype mediates vasodilation and is growth inhibitory. The studies of Munzenmaier and Greene (1996) indicate that both the AT 1 and the AT 2 angiotensin receptor subtypes are present in cremasteric arterioles. Therefore, the difference in the effect of high-salt diet on angiotensininduced constriction of skeletal muscle resistance arteries and cremasteric arterioles in the present study could be due, at least in part, to differences in the distribution of the AT 1 and AT 2 receptors and/or a differential regulation of the AT 1 and AT 2 receptor subtypes in these vessels. In the case of the cremasteric
arterioles, this could result in a shift in the balance of the vasoconstrictor (AT 1) and vasodilator (AT 2) receptors, which would prevent the enhanced constriction of the arterioles in response to angiotensin II. However, the effect of changes in dietary salt intake on the density of different angiotensin II receptor subtypes in the microcirculation and the ultimate effect of changes in these receptor subtypes on microvascular regulation remain to be completely determined. The latter questions appear to be an important area for future investigation. Another important finding of the present study was that short-term elevations of dietary salt intake had no effect on the response of either resistance arteries (Fig. 2B) or cremasteric arterioles (Fig. 3B) to angiotensin II. These findings contrast with the rapid blunting of the vascular relaxation in response to a variety of vasodilator stimuli that was demonstrated in resistance arteries and cremasteric arterioles after only 3 days on a high-salt diet (Weber et al., 1997; Frisbee and Lombard, 1998 1999). Thus, the potentiation of angiotensin IIinduced vasoconstriction of resistance arteries by elevated dietary salt intake takes considerably longer to develop than the impaired dilator responses of small arteries that were demonstrated in previous studies.
Responses to Norepinephrine In contrast to the potentiation of angiotensin IIinduced constriction of skeletal muscle resistance arteries by chronic elevations in dietary salt intake, neither chronic nor short-term elevations in dietary salt intake affected norepinephrine-induced constriction of the resistance arteries (Fig. 4) or the cremasteric arterioles (Fig. 5) in the present study. These findings are consistent with the results of a previous study by Nilsson et al. (1985), who reported no change in the norepinephrine sensitivity of the isolated perfused hindquarters preparation of WKY and SHR rats maintained on low-, normal, and high-salt diets for 8 –10 weeks. In another study, Yoshida et al. (1987) reported no difference in norepinephrine-induced constriction in aortic and renal artery strips of rabbits maintained on low-, normal, and high-sodium diets. In contrast, Soltis et al. (1993) reported that the vasoconstrictor response to norepinephrine was enhanced in thoracic
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aortic rings of rats that were maintained on a high-salt diet for 4 weeks after weaning and then returned to normal salt levels for 10 weeks. However, the latter study was confounded by an increase in systolic blood pressure in the high-salt diet rats, suggesting that the change in norepinephrine sensitivity in that study resulted from the increase in arterial blood pressure, rather than the elevation in dietary salt intake.
Myogenic Response In the present study, we also tested the effect of elevated dietary salt intake on the myogenic response of skeletal muscle resistance arteries to transmural pressure elevation. The myogenic response is an intrinsic, non-agonist-dependent contractile response that occurs in response to elevated pressure in many blood vessels. Myogenic responses allow blood vessels to increase their level of active tone in order to maintain a constant diameter or to constrict in response to increases in intralumenal pressure. This is an important mechanism for the local regulation of blood flow and downstream perfusion pressure. In the current studies, neither chronic nor short-term exposure to a high-salt diet affected the myogenic activation of the vessels. This finding is in contrast to previous studies by Takenaka et al. (1992), who reported an impaired myogenic response of renal afferent arterioles in the isolated hydronephrotic kidney preparation of Dahl R rats on a high-salt diet relative to controls on a low-salt diet. This may reflect differences in the strains of rats, the experimental preparation, or the function of these specific vessel types. For example, renal afferent arterioles are much smaller than the skeletal muscle resistance arteries used in the present study, and hence, they may be more sensitive to changes in intralumenal pressure or other mediators of local blood flow regulation.
SUMMARY AND CONCLUSIONS The current experiments have demonstrated that chronic, but not short-term, elevations in dietary salt intake lead to a selective potentiation of the response
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Weber, Frisbee, and Lombard
of skeletal muscle resistance arteries to angiotensin II. The increased sensitivity of the resistance arteries to angiotensin II may allow these vessels to adjust their tone in response to changes in the circulating levels of this peptide, even when plasma angiotensin II levels are reduced in response to the high-salt diet. This may be important in the regulation of resistance and blood flow within the vasculature. The lack of a change in the sensitivity of cremasteric arterioles to angiotensin II may be a protective mechanism of the microvasculature to maintain tissue blood flow and nutrient delivery despite the reductions in microvessel density that occur due to microvascular rarefaction associated with the high-salt diet (Greene et al., 1990; HansenSmith et al., 1996). The lack of a change in the response of the resistance arteries to norepinephrine and transmural pressure elevation and the lack of an enhanced response of the cremasteric arterioles to norepinephrine suggest that alterations in vasoconstrictor responses during chronic elevations in dietary salt intake are not generalized, but are specific to vasoconstrictor stimuli that are sensitive to changes in dietary salt levels.
ACKNOWLEDGMENTS This study was supported by NIH Grants HL 29587, HL 37374, and HL 52211. The authors thank Kathy Valent, Jane Schimke, and Barbara Vasko for their fine secretarial assistance.
REFERENCES Aguilera, G., and Catt, K. (1981). Regulation of vascular angiotensin II receptors in the rat during altered sodium intake. Circ. Res. 49, 751–758. Boegehold, M. A. (1993a). Microvascular changes associated with high salt intake and hypertension in Dahl rats. Int. J. Microcirc. Clin. Exp. 12, 143–156. Boegehold, M. A. (1993b). Effect of dietary salt on arteriolar nitric oxide in striated muscle of normotensive rats. Am. J. Physiol. 264, H1810 –H1816. Catt, K. J., Mendelsohn, F. A. C., Millan, M. A., and Aguilera, G. (1984). The role of angiotensin II receptors in vascular regulation. J. Cardiovasc. Pharmacol. 6, S575–S586.
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Fredricks, K. T., Liu, Y., and Lombard, J. H. (1994). Response of extraparenchymal resistance arteries of rat skeletal muscle to reduced PO 2. Am. J. Physiol. 267, H706 –H715. Frisbee, J. C., and Lombard, J. H. (1998). Chronic elevations in salt intake and reduced renal mass hypertension compromise mechanisms of arteriolar dilation. Microvasc. Res. 56, 218 –227. Frisbee, J. C., and Lombard, J. H. (1999). Acute elevations in salt intake and reduced renal mass hypertension compromise mechanisms of arteriolar dilation. Microvasc. Res., in press. Greene, A. S., Lombard, J. H., Cowley, A. W., Jr., and Hansen-Smith, F. M. (1990). Microvessel changes in hypertension measured by Griffonia simplicifolia I lectin. Hypertension 15, 779 –783. Hansen-Smith, F. M., Greene, A. S., Cowley, A. W., Jr., and Lombard, J. H. (1990). Structural changes during microvascular rarefaction in chronic hypertension. Hypertension 15, 922–928. Hansen-Smith, F. M., Morris, L. W., Greene, A. S., and Lombard, J. H. (1996). Rapid microvessel rarefaction with elevated salt intake and reduced renal mass hypertension in rats. Circ. Res. 79, 324 –330. Liu, Y., Fredricks, K. T., Roman, R. J., and Lombard, J. H. (1997). Response of resistance arteries to reduced PO 2 and vasodilators during hypertension and elevated salt intake. Am. J. Physiol. 273, H869 –H877. Lombard, J. H., Hinojosa-Laborde, C., and Cowley, A. W. (1989). Hemodynamics and microcirculatory alterations in reduced renal mass hypertension. Hypertension 13, 128 –138. Meininger, G. A., and Davis, M. J. (1992). Cellular mechanisms involved in the vascular myogenic response. Am. J. Physiol. 263, H647–H659. Munzenmaier, D. H., and Greene, A. S. (1996). Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension 27 (Part 2), 760 –765. Nilsson, H., Ely, D., Friberg, P., Karlstrom, G., and Folkow, B. (1985). Effects of high and low sodium diets on the resistance vessels and
319 their adrenergic vasoconstrictor fibre control in normotensive (WKY) and hypertensive (SHR) rats. Acta Physiol. Scand. 125, 323–334. Rieder, M. J., Roman, R. J., and Greene, A. S. (1997). Reversal of microvascular rarefaction and reduced renal mass hypertension. Hypertension 30 (Part 1), 120 –127. Scheuer, D. A., and Perrone, M. H. (1993). Angiotensin type 2 receptors mediate depressor phase of biphasic pressure response to angiotensin. Am. J. Physiol. 264, R917–R923. Soltis, E. E., Bartrug, B. L., and Newman, P. S. (1993). Chronic hypertension and vascular alterations following short-term high dietary salt intake in postweaning rats. Dev. Pharmacol. Ther. 20, 107–110. Stoll, M., Meffert, S., Stroth, U., and Unger, T. (1995a). Growth or antigrowth: Angiotensin and the endothelium. J. Hypertens. 13, 1529 –1534. Stoll, M., Steckelings, U., Paul, M., Bottari, S. P., Metzger, R., and Unger, T. (1995b). The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J. Clin. Invest. 95, 651– 657. Takenaka, T., Forster, H., De Micheli, A., and Epstein, M. (1992). Impaired myogenic responsiveness of renal microvessels in dahl salt-sensitive rats. Circ. Res. 71, 471– 480. Unger, T., Chung, O., Csikos, T., Culman, J., Gallinat, S., Gohlke, P., Hohle, S., Meffert, S., Stoll, M., Stroth, U., and Zhu, Y. (1996). Angiotensin receptors. J. Hypertens. 14(Suppl. 5), S95–S103. Weber, D. S., Greene, A. S., and Lombard, J. H. (1997). Effect of short term and chronic high salt diet on vasodilator response of skeletal muscle resistance arteries. FASEB J. 11, A43. [Abstract] Yoshida, M., Ueda, S., Machida, J., and Ikegami, K. (1987). The change of vascular reactivity to angiotensin II and norepinephrine in the two-kidney, one-clip renovascular hypertensive rabbit. J. Urol. 137, 1048 –1052.
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