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Acute Elevations in Salt Intake and Reduced Renal Mass Hypertension Compromise Arteriolar Dilation in Rat Cremaster Muscle
Microvascular Research 57, 273–283 (1999) Article ID mvre.1998.2138, available online at http://www.idealibrary.com on
Acute Elevations in Salt Intake and Reduced Renal Mass Hypertension Compromise Arteriolar Dilation in Rat Cremaster Muscle Jefferson C. Frisbee and Julian H. Lombard Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received July 6, 1998
Alterations in arteriolar reactivity to dilator agonists were assessed in the skeletal muscle microcirculation of normotensive male Sprague–Dawley rats fed either high(4% NaCl; HS) or low- (0.4% NaCl; LS) salt diets and in reduced renal mass hypertensive rats (RRM-HT) on a high-salt diet for 3 days. An in situ cremaster muscle preparation was superfused with physiological salt solution, transilluminated, and viewed via television microscopy. A videomicrometer was used to measure changes in diameter of distal arterioles in response to increasing concentrations of acetylcholine (ACH), iloprost (ILO), cholera toxin (CT), forskolin (FOR), and sodium nitroprusside (SNP). Arteriolar dilation in response to ACH, ILO, and CT was significantly reduced in both HS and RRM-HT rats, while responses to FOR and SNP were decreased in RRM-HT rats only. The maximum dilation of the arterioles (determined during superfusion of the muscle with Ca 21-free solution containing 10 24 M adenosine) was similar in the normotensive control animals on LS and HS diets, but was reduced in the RRM-HT rats, suggesting that early anatomic remodeling of the vessel wall may be occurring with RRM-HT. We conclude that arteriolar reactivity to endothelium-dependent and -independent vasodilator agonists is impaired as early as 3 days after the development of RRM hypertension or commencement of a high-salt diet in normotensive rats. Structural remodeling of the arteriolar wall, although becoming evident in the hypertensive rats, takes longer 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
to develop than the impaired vasodilator reactivity. © 1999 Academic Press
Key Words: hypertension; salt; microcirculation; vascular reactivity; angiotensin II; vasodilation; vascular smooth muscle.
INTRODUCTION Previous studies have demonstrated that both hypertension (Stacy and Prewitt, 1989; Ono et al., 1989; Vicaut, 1992; Struijker Boudier et al., 1992; Stekiel et al., 1993; Izzard and Heagerty, 1995; Hernandez and Greene, 1995; Rachev et al., 1996) and chronic elevations in dietary salt intake in normotensive animals (Boegehold and Kotchen, 1990; Hernandez et al., 1992; Boegehold, 1993a,b; Frisbee and Lombard, 1998) cause significant alterations in the structure and function of skeletal muscle microvessels. Chronic reduced renal mass (RRM) hypertension and chronic elevations in dietary salt intake in normotensive animals lead to structural alterations in microvessels which have the potential to physically constrain vascular relaxation in response to dilator stimuli (Frisbee and Lombard, 1998; Hansen-Smith et al., 1990, 1991; Boegehold, 1993b). While it has been assumed that these alterations are slow to develop, several studies have indicated that alterations in vascular
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structure can occur very rapidly, even preceding the elevations in blood pressure (Rorive et al., 1980; Loeb et al., 1986; Hansen-Smith et al., 1996). With regard to microvessel function, studies performed both in our laboratory (Liu et al., 1997; Frisbee and Lombard, 1998) and by others (Stekiel et al., 1993; Boegehold, 1993a; Carvalho et al., 1997; Houghton et al., 1998) have identified decreased vasodilator reactivity in human and rat muscle during high-salt diet in normotensive animals and in different models of hypertension. Of greatest relevance to the present studies, vessel responses to dilator stimuli acting at the vascular endothelium and the vascular smooth muscle membrane receptors become compromised in rat skeletal muscle resistance arteries (Liu et al., 1997) and arterioles (Frisbee and Lombard, 1998) during chronic high-salt diet and RRM hypertension, compared to vasodilator responses in normotensive rats on a lowsalt diet. Frisbee and Lombard (1998) also demonstrated a reduced arteriolar reactivity to agonists acting at the heterotrimeric G proteins and the intracellular second-messenger systems in rats with RRM hypertension. However, there has been no attempt to determine the effect of short-term high-salt diet (either in conjunction with or independent of hypertension) on the reactivity of the skeletal muscle arterioles to vasodilator stimuli. The present experiments tested the hypothesis that acute elevations in dietary salt intake in normotensive animals and RRM-hypertensive rats lead to an impaired relaxation in response to vasodilator stimuli in the cremaster muscle microcirculation. These experiments also sought to identify potential mechanisms that may be responsible for impaired arteriolar relaxation in acute hypertension and during short-term exposure to a high-salt diet, by utilizing vasodilator agonists that act on different sites in the membrane receptor–signal transduction pathway. These include: (1) the endothelium-dependent vasodilator acetylcholine; (2) the stable prostacyclin analog iloprost, which acts directly on receptors on the smooth muscle cell membrane to cause vasodilation via the cyclic AMP pathway; (3) the G S protein activator cholera toxin; (4) the endothelium-independent nitric oxide donor sodium nitroprusside; and (5) the direct adenylyl cyclase activator forskolin. Finally, the
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Frisbee and Lombard
present experiments also sought to determine the effects of short-term hypertension and elevated salt intake on the maximum vessel diameter, as an indication of the extent to which structural remodeling of arterioles may impact upon the ability of the vessel to increase its diameter when the smooth muscle is relaxed.
MATERIALS AND METHODS Renal mass reduction. Male Sprague–Dawley rats were anesthetized with a 9:2 mixture of 100 mg z ml 21 ketamine and 10 mg z ml 21 acepromazine (0.1 ml z 100 g 21 body weight). The animals were prepared by exposing the left kidney via a flank incision, followed by removal of the superior and inferior poles of the kidney to reduce its total mass by approximately 50% (Lombard et al., 1989). After the remainder of the kidney was returned to the abdominal cavity and the incision was closed, penicillin (40,000 units) was injected into the muscles of the hindlimb to prevent postoperative infection. After a 2-week recovery period, the right kidney was removed, resulting in a final reduction of approximately 75% of the animal’s total renal mass. After an additional week of recovery, these RRM rats were placed on a high-salt (4.0% NaCl) diet (Dyets, Bethlehem, PA) for 3 days, to produce the RRM hypertension. Animal groups and preparation. Vasodilator reactivity studies were conducted on the RRM-hypertensive rats and in two groups of normotensive Sprague– Dawley controls, summarized in Table 1. In the present experiments, normotensive control rats not undergoing the RRM procedure were maintained on either a low-salt diet (0.4% NaCl, Dyets) or the highsalt diet for 3 days. In preliminary studies in our laboratory, we determined that sham-operated rats on high- and low-salt diets, serving as normotensive control animals for RRM-hypertensive rats, had dilator responses that were similar to those of rats that were on the high- and low-salt diets, but not subjected to the sham surgery. All rats were allowed to drink tap water, ad libitum. Body weights for normotensive rats maintained on either the low- or the high-salt diet
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were not different, while body weight in the RRMhypertensive group was significantly less than that of the normotensive controls on the low- or high-salt diet (Table 1). Methods and protocols for microcirculatory studies. On the day of the experiment, individual rats were anesthetized with an injection (60 mg z kg 21, ip) of pentobarbital sodium (Veterinary Laboratories, Lenexa, KS), and the trachea was cannulated to ensure a patent airway. The carotid artery and external jugular vein were cannulated for arterial pressure recording and for 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 (PSS), 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 2PO 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). The vessel selection procedure for these experiments was as follows: In a clearly visible region of the cremaster muscle, a second-order arteriole of approximately 60-mm diameter was identified. This vessel was tracked along its length, and successive branches were identified up to the point at which capillaries arose from the terminal arterioles. Once the capillaries arising from this arteriolar network were identified, the section of the arteriole that was immediately proximal to the capillaries was selected for analysis. In this selection procedure, the location of the arterioles (immediately proximal to the capillaries) was the selection criterion, and the absolute diameter of the arteriole was not a factor. At this point, the ultimate selection of the arterioles was subject to the following additional requirements: (1) location in a region of the muscle that was away from any incision, (2) clearly discernible vessel walls, (3) brisk flow velocity, and (4) active tone, as indicated by the occurrence of a brisk dilation in response to topical application of 10 24 M adenosine.
Arteriolar diameters were measured before and after the topical application of each of the following agents to the preparation: (1) acetylcholine (10 28–10 26 M), (2) iloprost (10 215–10 29 g z ml 21), (3) cholera toxin (10 29 g z ml 21), (4) sodium nitroprusside (10 28–10 26 M), and (5) forskolin (10 213–10 27 M). The maximum arteriolar diameter was also assessed in all arterioles by measuring the vessel response to superfusion with calcium-free physiological salt solution containing 10 24 M adenosine. Arteriolar diameters were measured after the vasodilator response to the individual agonists had stabilized, which occurred within 20 s of the agonist challenge. Successive agonist challenges were applied only after the vessel had returned to its resting diameter following the application of the preceding agonist. The application of the agents was randomized to prevent the possible occurrence of ordering effects and to control for any time-dependent changes in vascular reactivity during the experiment. The total experiment duration was approximately 150 min, and viability of the preparation was assessed by verifying that the vessels maintained normal levels of resting tone throughout the experiment. Data and statistical analysis. All data are presented as the mean 6 SEM of either the absolute vessel diameter (mm) or the change in diameter from rest in response to the agonist challenge (%). Analysis of variance was employed to determine initial differences across experimental conditions and Scheffe’s post hoc test was employed to identify significant differences between specific experimental groups. In all cases, a probability level of P , 0.05 was considered statistically significant.
RESULTS Mean arterial pressure and resting arteriolar diameter. Mean arterial pressure (MAP) and resting diameter of the cremasteric arterioles in the three experimental groups are summarized in Table 1. MAP was significantly elevated in RRM-hypertensive rats compared to either normotensive group. MAP in normotensive animals on the high-salt and low-salt diet was
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TABLE 1 Experimental Groups for Arteriolar Dilator Reactivity Studies Group
n
Age (weeks)
Mass (g)
MAP (mm Hg)
Diet
Normotensive Normotensive Hypertensive
12 12 12
12–13 12–13 12–13
356 6 18 362 6 19 318 6 12*†
119 6 4.6 123 6 3.4 139 6 3.3*†
LS HS HS
Rest diameter (mm) 18.0 6 1.0 18.8 6 1.0 19.3 6 0.9
Note. LS represents low-salt-content rat chow (0.4% NaCl), HS represents high-salt-content rat chow (4.0% NaCl). Asterisks represent significant differences between the age-matched groups compared to the normotensive controls on the low-salt diet. Daggers represent significant differences from normotensive controls on the high-salt diet.
not different. Resting diameter of the arterioles, prior to agonist application, was not significantly different across the three experimental groups. Responses to acetylcholine. Figure 1 summarizes the arteriolar responses to acetylcholine across the experimental groups. Arterioles of rats on the low-salt diet exhibited a significantly greater dilation than those of the normotensive controls on the high-salt
diet or the RRM-hypertensive rats. The dilation of arterioles in response to the higher concentrations of acetylcholine was also significantly greater in normotensive rats on the high-salt diet than in the RRMhypertensive animals. Responses to iloprost. Arteriolar responses to iloprost are summarized in Fig. 2. There were no significant differences in the response of arterioles to ilo-
FIG. 1. Responses of cremasteric arterioles to increasing concentrations of acetylcholine in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a LS diet; daggers indicate a significant reduction in the dilation compared to normotensive controls on a HS diet.
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FIG. 2. Responses of cremasteric arterioles to increasing concentrations of iloprost in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a LS diet; daggers indicate a significant reduction in the dilation compared to normotensive controls on a HS diet.
prost in the normotensive rats on low- and high-salt diets, except for the dilation in response to 10 29 g z ml 21, in which the arterioles of normotensive rats on the high-salt diet dilated significantly less than those of normotensive controls on a low-salt diet. In the RRM-hypertensive rats, arteriolar dilation in response to iloprost was significantly reduced relative to normotensive controls on either the low- or the highsalt diet. Responses to cholera toxin. In the animals on the low-salt diet, challenge with cholera toxin (10 29 g z ml 21) resulted in a 28 6 4.5% increase in the diameter of the cremasteric arterioles above their resting value (Fig. 3). This response was significantly larger than that of either the normotensive rats fed the highsalt diet (18 6 2.7%) or the RRM-hypertensive rats (12 6 1.3%). Responses to forskolin. Figure 4 summarizes the response of cremasteric arterioles to the topical appli-
cation of forskolin in the three experimental groups. There were no significant differences in the response of the arterioles to forskolin in normotensive animals on low-salt and high-salt diets. However, arteriolar responses to forskolin were significantly reduced in RRM-hypertensive animals compared to those in normotensive controls on the low- and high-salt diets. Responses to sodium nitroprusside. Arteriolar responses to sodium nitroprusside are summarized in Fig. 5. The vessel responses to nitroprusside were not significantly different in normotensive animals on low- and high-salt diets. However, arteriolar reactivity to nitroprusside was significantly decreased in the RRM-hypertensive rats compared to the normotensive controls on either the low-salt or the high-salt diet. Maximal dilation in response to calcium-free superfusate plus adenosine. Figure 6 summarizes the response of the cremasteric arterioles to superfusion with calcium-free PSS containing 10 24 M adenosine in
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FIG. 3. Responses of cremasteric arterioles to cholera toxin (10 29 g z ml 21) in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a low-salt diet.
LS, HS, and HSRRM rats. In these experiments, the maximum dilation from rest was not different in the normotensive rats on low-salt and high-salt diets, but was significantly reduced in the RRM-hypertensive rats.
DISCUSSION Recent work in our laboratory has demonstrated that a chronic elevation in dietary salt intake in normotensive rats and in RRM-hypertensive rats impairs the reactivity of skeletal muscle resistance arteries and distal arterioles to dilator agonists compared to responses in vessels from normotensive rats on low-salt
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diets. These altered reactivity patterns have been identified using an array of dilator agonists, each exerting its effect at either the endothelium or the vascular smooth muscle, and at different sites in the signal transduction pathway (Liu et al., 1997; Frisbee and Lombard, 1998). Preliminary investigations of the time course of the altered reactivity in skeletal muscle resistance arteries of normotensive rats during elevated dietary salt intake demonstrated that vessel responses to acetylcholine and iloprost are reduced compared to those of rats on a low-salt diet after as little as 3 days on the high-salt diet (Weber et al., 1997). In addition, Hansen-Smith et al. (1996) demonstrated that structural alterations in microvessels can occur over this same 3-day period in both RRM-hypertensive rats and normotensive animals on a high-salt diet. Taken to-
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FIG. 4. Responses of cremasteric arterioles to increasing concentrations of forskolin in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a LS diet; daggers indicate a significant reduction in the dilation compared to normotensive controls on a HS diet.
gether, these observations indicate that vascular adaptations to elevated salt intake and RRM hypertension may occur very rapidly. The present study builds on these observations to determine if the relaxation of cremasteric arterioles in response to endothelium-dependent and -independent dilator agonists is altered over a 3-day period in RRM-hypertensive rats and in normotensive rats on a high-salt diet, relative to normotensive controls on a low-salt diet. A further goal of these experiments was to determine whether structural remodeling of the arterioles, which would limit the ability of the vessels to increase their diameter in response to dilator stimuli, could contribute to an impaired relaxation of arterioles in response to vasodilator agonists in RRMhypertensive rats or in normotensive animals subjected to short-term increases in dietary salt intake. Responses to vasodilator agonists. Interpretation of the patterns of arteriolar reactivity in response to the agonist challenges in this study indicated a clear
trend for a decreased responsiveness to the endothelium-dependent agonist acetylcholine, the smooth muscle receptor-dependent agonist iloprost, and the heterotrimeric G-protein activator cholera toxin in normotensive rats on the high-salt diet and in RRMhypertensive rats, compared to the normotensive controls on a low-salt diet. While similar patterns of impaired vasodilator reactivity in cremasteric arterioles have been previously identified over a chronic (4week) time frame in RRM-hypertensive rats and in normotensive control animals on a high-salt diet (Frisbee and Lombard, 1998), the present study presents the first observations demonstrating conclusively that the impaired vascular reactivity in RRM-hypertensive rats and normotensive rats on a high-salt diet develops in as little as 3 days. A crucial question regarding the impaired vasodilator responses in the hypertensive rats and in the normotensive rats on high-salt diet is the mechanism of the impaired relaxation. Specifically, is the reduced
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FIG. 5. Responses of cremasteric arterioles to increasing concentrations of sodium nitroprusside in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a low-salt diet.
dilator response due to an impaired function of the mechanisms mediating active relaxation of the vascular smooth muscle, to structural alterations in the arteriolar wall that impair the ability of the vessel to dilate, or to both? This question is of added interest, given the very short time period (3 days) over which impaired vasodilator responses develop, which is much more rapid than previously established. If the reduced dilation of arterioles in response to the different vasodilator agents in RRM-hypertensive rats and in normotensive controls on a high-salt diet is due to alterations in active vasodilator mechanisms, this could have resulted from compromised function at several sites in the transduction pathway. This conclusion is based on the observation that arteriolar relaxation was impaired during the application of several dilator agents with different mechanisms of action. These include acetylcholine, which acts on receptors on the vascular endothelium; iloprost, which
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acts directly on receptors on the vascular smooth muscle cell membrane; and cholera toxin, which activates the G S protein leading to relaxation of cremasteric arterioles (Stekiel et al., 1993). Alternatively, this general impairment of active vessel relaxation in response to a wide variety of vasodilator stimuli may reflect a defect at a common point in the signal transduction pathway that is downstream of the site of action of the various vasodilator agonists, e.g., defective K 1 channel regulation, defective regulation of intracellular Ca 21 levels, etc. The hypothesis that the mechanisms responsible for active vasodilation are impaired in arterioles of animals with RRM hypertension is consistent with the results of Stekiel et al. (1993), who reported that stimulation of b-adrenergic receptors of first-order arterioles with isoproterenol and activation of G proteins with cholera toxin had no effect on the transmembrane potential of arteriolar smooth muscle cells in RRM-
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FIG. 6. Maximal dilation of cremasteric arterioles in response to superfusion with Ca 21-free PSS containing 10 24 M adenosine in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a low-salt diet.
hypertensive rats, but hyperpolarized the vascular smooth muscle in normotensive controls. Those observations indicated that the b-receptor-mediated cascade leading to smooth muscle hyperpolarization and relaxation was depressed with chronic RRM hypertension, and suggested that the specific site of this depression may have been at the G S protein–adenylyl cyclase coupling stage. In addition, preliminary studies by our group indicate that a short-term (3 days) elevation of dietary salt intake eliminates vascular smooth muscle hyperpolarization and vascular relaxation in middle cerebral arteries of normotensive rats (Lombard et al., 1997), which is also consistent with the interpretation that some of the mechanisms mediating active relaxation of the vessels in response to different vasodilator stimuli are impaired by exposure to a high-salt diet.
Structural remodeling in arterioles of rats on a highsalt diet. An alternative interpretation of the results of the present experiments is that the diminished relaxation of vessels of the RRM-hypertensive rats and the normotensive rats on the high-salt diet in response to the agonist challenges is due to structural alterations in the arteriolar wall of these animals, which could physically hinder the ability of the vessels to increase their diameter, even in the absence of defects in active vasodilator mechanisms. The interpretation that structural alterations can occur rapidly in rats with acute RRM hypertension is consistent with reports in the literature (Rorive et al., 1980; Loeb et al., 1986; Hansen-Smith et al., 1996) and with our present observation of a significant reduction in the response of cremasteric arterioles to superfusion with Ca 21-free PSS plus adenosine in hypertensive animals. This re-
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duced response to Ca 21-free PSS plus adenosine may be indicative of the earliest stages of structural remodeling of the arterioles in animals with RRM hypertension. In contrast, there was no evidence in the present study for structural remodeling of arterioles in response to acute elevations in dietary salt intake alone. However, in our earlier study, vascular remodeling was identified in normotensive rats after a chronic elevation of dietary salt intake (Frisbee and Lombard, 1998). Taken together, these observations suggest that structural changes in normotensive rats on a high-salt diet develop more slowly in the absence of the elevated blood pressure that is associated with the development of hypertension. However, the relative magnitude of the relaxation of the arterioles in response to the various vasodilator agonists used in the present study suggests that it is unlikely that structural remodeling was the sole factor leading to the impaired relaxation of the vessels of the hypertensive rats and normotensive animals on the high-salt diet. In these experiments, the magnitudes of the maximal dilation of the cremasteric arterioles during superfusion with Ca 21-free PSS containing 10 24 M adenosine in normotensive rats on the high-salt diet and in RRM-hypertensive rats (71.8 6 12.1 and 59.1 6 6.5% above the initial rest vessel diameter, respectively) were much greater than the increase in vessel diameter in response to any of the dilator agonists in the present study. This observation suggests that an inability of the vessel to increase its diameter due to structural remodeling was not the only factor responsible for the reduced dilation in response to the agonists used in this study. However, it is conceivable that structural alterations could reduce the distensibility of arterioles in animals with acute RRM hypertension and in animals subjected to short-term elevations in dietary salt intake, thereby interfering with the ability of the vessels to increase their diameter following relaxation of the vascular smooth muscle. Potential mechanisms for the altered microvessel structure and function. Previous studies have suggested that the renin–angiotensin system may play a critical role in the maintenance of the normal structure of the microcirculation and that this protective effect may be lost when circulating angiotensin II is suppressed during RRM hypertension and elevated di-
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Frisbee and Lombard
etary salt intake (Hernandez et al., 1992; Rieder et al., 1997). Preliminary investigations in our laboratory have suggested that the suppression of the renin– angiotensin system with high-salt diet may also play a critical role in contributing to the impaired vascular reactivity in rat skeletal muscle resistance arteries (Weber et al., 1997) and in rat middle cerebral arteries (Lombard et al., 1997). In those studies, chronic intravenous infusion of a low dose of angiotensin II in rats maintained on a high-salt diet restored the normal vasodilator reactivity that was suppressed in arteries of animals on a high-salt diet that did not receive angiotensin II infusion. Taken together, these experiments suggest that angiotensin II suppression may be a critical element regulating the alterations in microvessel structure and function that develop with RRM hypertension and high-salt diet. Conclusions. When considered in combination with our existing work (Frisbee and Lombard, 1998; Weber et al., 1997; Liu et al., 1997; Lombard et al., 1997) and the findings of other studies using the one-kidney, one-clip model of hypertension (Hashimoto et al., 1987), the observations reported in the present study can begin to be integrated into a conceptual model addressing microvascular adaptations to the development of RRM hypertension and elevations in salt intake. It is evident that, with the development of hypertension, the ability of skeletal muscle resistance arteries and arterioles to relax in response to vasodilator stimuli is rapidly impaired. Structural alterations appear to develop at a slower rate and occur earlier in hypertensive animals than in normotensive controls. As the duration of the RRM hypertension and the period of elevated salt intake is increased, the remodeling becomes more extensive, and the anatomic adaptations become clearly evident both during chronic elevations of salt intake in normotensive animals and in RRM hypertension. The mechanisms and interplay between these physiological and anatomic processes, the potential for reversibility of the changes in structure and reactivity, the role of the renin–angiotensin system in contributing to these changes, and the functional implications of these physiological and anatomical changes remain to be identified and represent exciting areas for future investigation.
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ACKNOWLEDGMENTS This work was supported by NIH Grants HL29587, HL37374, and HL52211. The present studies were reported in preliminary form at the 1998 Microcirculatory Society meetings in San Francisco, California. The authors thank the Berlex Corporation for their generous donation of the iloprost used in these experiments.
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283 Houghton, J. L., Davison, C. A., Kuhner, P. A., Torossov, M. T., Strogatz, D. S., and Carr, A. A. (1998). Heterogeneous vasomotor responses of coronary conduit and resistance vessels in hypertension. J. Am. Coll. Cardiol. 31, 374 –382. Izzard, A. S., and Heagerty, A. M. (1995). Hypertension and the vasculature: Arterioles and the myogenic response. J. Hypertens. 13, 1– 4. 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. Loeb, A. L., Mandel, H. G., Straw, J. A., and Bean, B. L. (1986). Increased aortic DNA synthesis precedes renal hypertension in rats. An obligatory step? Hypertension 8, 754 –761. Lombard, J. H., Bizub, D., Weber, D. S., and Greene, A. S. (1997). Short term elevation in salt intake impairs electrical and mechanical responses of rat middle cerebral arteries to reduced PO 2 and prostacyclin via suppression of angiotensin II (AngII). Microcirculation 4, 159. [Abstract] Lombard, J. H., Hinojosa-Laborde, C., and Cowley, A. W. (1989). Hemodynamics and microcirculatory alterations in reduced renal mass hypertension. Hypertension 13, 128 –138. Ono, Z., Prewitt, R. L., and Stacy, D. L. (1989). Arteriolar changes in developing and chronic stages of two-kidney, one clip hypertension. Hypertension 14, 36 – 43. Rachev, A., Stergiopulos, N., and Meister, J. J. (1996). Theoretical study of dynamics of arterial wall remodeling in response to changes in blood pressure. J. Biomech. 29, 635– 642. Rieder, M. J., Roman, R. J., and Greene, A. S. (1997). Reversal of microvascular rarefaction and reduced renal mass hypertension. Hypertension 30, 120 –127. Rorive, G. L., Carlier, P. J., and Foidart, J. M. (1980). Hyperplasia of rat arteries smooth muscle cells associated with the development and reversal of renal hypertension. Clin. Sci. 59(Suppl. 6), 335s– 338s. Stacy, D. L., and Prewitt, R. L. (1989). Effects of chronic hypertension and its reversal on arteries and arterioles. Circ. Res. 65, 869 – 879. Stekiel, W. J., Contney, S. J., and Rusch, N. J. (1993). Altered betareceptor control of in situ membrane potential in hypertensive rats. Hypertension 21, 1005–1009. Struijker Boudier, H. A. J., le Noble, J. L. M. L., Messing, M. W. J., Huijberts, M. S. P., le Noble, F. A. C., and Van Essen, H. (1992). The microcirculation and hypertension. J. Hypertens. 10, 147–156. Vicaut, E. (1992). Hypertension and the microcirculation: A brief overview of experimental studies. J. Hypertens. 10, S59 –S68. Weber, D. S., Greene, A. S., and Lombard, J. H. (1997). Effect of short term and chronic high salt diet on vasodilator responses of skeletal muscle resistance arteries. FASEB J. 11, A43. [Abstract]
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Acute Elevations in Salt Intake and Reduced Renal Mass Hypertension Compromise Arteriolar Dilation in Rat Cremaster Muscle Jefferson C. Frisbee and Julian H. Lombard Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received July 6, 1998
Alterations in arteriolar reactivity to dilator agonists were assessed in the skeletal muscle microcirculation of normotensive male Sprague–Dawley rats fed either high(4% NaCl; HS) or low- (0.4% NaCl; LS) salt diets and in reduced renal mass hypertensive rats (RRM-HT) on a high-salt diet for 3 days. An in situ cremaster muscle preparation was superfused with physiological salt solution, transilluminated, and viewed via television microscopy. A videomicrometer was used to measure changes in diameter of distal arterioles in response to increasing concentrations of acetylcholine (ACH), iloprost (ILO), cholera toxin (CT), forskolin (FOR), and sodium nitroprusside (SNP). Arteriolar dilation in response to ACH, ILO, and CT was significantly reduced in both HS and RRM-HT rats, while responses to FOR and SNP were decreased in RRM-HT rats only. The maximum dilation of the arterioles (determined during superfusion of the muscle with Ca 21-free solution containing 10 24 M adenosine) was similar in the normotensive control animals on LS and HS diets, but was reduced in the RRM-HT rats, suggesting that early anatomic remodeling of the vessel wall may be occurring with RRM-HT. We conclude that arteriolar reactivity to endothelium-dependent and -independent vasodilator agonists is impaired as early as 3 days after the development of RRM hypertension or commencement of a high-salt diet in normotensive rats. Structural remodeling of the arteriolar wall, although becoming evident in the hypertensive rats, takes longer 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
to develop than the impaired vasodilator reactivity. © 1999 Academic Press
Key Words: hypertension; salt; microcirculation; vascular reactivity; angiotensin II; vasodilation; vascular smooth muscle.
INTRODUCTION Previous studies have demonstrated that both hypertension (Stacy and Prewitt, 1989; Ono et al., 1989; Vicaut, 1992; Struijker Boudier et al., 1992; Stekiel et al., 1993; Izzard and Heagerty, 1995; Hernandez and Greene, 1995; Rachev et al., 1996) and chronic elevations in dietary salt intake in normotensive animals (Boegehold and Kotchen, 1990; Hernandez et al., 1992; Boegehold, 1993a,b; Frisbee and Lombard, 1998) cause significant alterations in the structure and function of skeletal muscle microvessels. Chronic reduced renal mass (RRM) hypertension and chronic elevations in dietary salt intake in normotensive animals lead to structural alterations in microvessels which have the potential to physically constrain vascular relaxation in response to dilator stimuli (Frisbee and Lombard, 1998; Hansen-Smith et al., 1990, 1991; Boegehold, 1993b). While it has been assumed that these alterations are slow to develop, several studies have indicated that alterations in vascular
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structure can occur very rapidly, even preceding the elevations in blood pressure (Rorive et al., 1980; Loeb et al., 1986; Hansen-Smith et al., 1996). With regard to microvessel function, studies performed both in our laboratory (Liu et al., 1997; Frisbee and Lombard, 1998) and by others (Stekiel et al., 1993; Boegehold, 1993a; Carvalho et al., 1997; Houghton et al., 1998) have identified decreased vasodilator reactivity in human and rat muscle during high-salt diet in normotensive animals and in different models of hypertension. Of greatest relevance to the present studies, vessel responses to dilator stimuli acting at the vascular endothelium and the vascular smooth muscle membrane receptors become compromised in rat skeletal muscle resistance arteries (Liu et al., 1997) and arterioles (Frisbee and Lombard, 1998) during chronic high-salt diet and RRM hypertension, compared to vasodilator responses in normotensive rats on a lowsalt diet. Frisbee and Lombard (1998) also demonstrated a reduced arteriolar reactivity to agonists acting at the heterotrimeric G proteins and the intracellular second-messenger systems in rats with RRM hypertension. However, there has been no attempt to determine the effect of short-term high-salt diet (either in conjunction with or independent of hypertension) on the reactivity of the skeletal muscle arterioles to vasodilator stimuli. The present experiments tested the hypothesis that acute elevations in dietary salt intake in normotensive animals and RRM-hypertensive rats lead to an impaired relaxation in response to vasodilator stimuli in the cremaster muscle microcirculation. These experiments also sought to identify potential mechanisms that may be responsible for impaired arteriolar relaxation in acute hypertension and during short-term exposure to a high-salt diet, by utilizing vasodilator agonists that act on different sites in the membrane receptor–signal transduction pathway. These include: (1) the endothelium-dependent vasodilator acetylcholine; (2) the stable prostacyclin analog iloprost, which acts directly on receptors on the smooth muscle cell membrane to cause vasodilation via the cyclic AMP pathway; (3) the G S protein activator cholera toxin; (4) the endothelium-independent nitric oxide donor sodium nitroprusside; and (5) the direct adenylyl cyclase activator forskolin. Finally, the
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Frisbee and Lombard
present experiments also sought to determine the effects of short-term hypertension and elevated salt intake on the maximum vessel diameter, as an indication of the extent to which structural remodeling of arterioles may impact upon the ability of the vessel to increase its diameter when the smooth muscle is relaxed.
MATERIALS AND METHODS Renal mass reduction. Male Sprague–Dawley rats were anesthetized with a 9:2 mixture of 100 mg z ml 21 ketamine and 10 mg z ml 21 acepromazine (0.1 ml z 100 g 21 body weight). The animals were prepared by exposing the left kidney via a flank incision, followed by removal of the superior and inferior poles of the kidney to reduce its total mass by approximately 50% (Lombard et al., 1989). After the remainder of the kidney was returned to the abdominal cavity and the incision was closed, penicillin (40,000 units) was injected into the muscles of the hindlimb to prevent postoperative infection. After a 2-week recovery period, the right kidney was removed, resulting in a final reduction of approximately 75% of the animal’s total renal mass. After an additional week of recovery, these RRM rats were placed on a high-salt (4.0% NaCl) diet (Dyets, Bethlehem, PA) for 3 days, to produce the RRM hypertension. Animal groups and preparation. Vasodilator reactivity studies were conducted on the RRM-hypertensive rats and in two groups of normotensive Sprague– Dawley controls, summarized in Table 1. In the present experiments, normotensive control rats not undergoing the RRM procedure were maintained on either a low-salt diet (0.4% NaCl, Dyets) or the highsalt diet for 3 days. In preliminary studies in our laboratory, we determined that sham-operated rats on high- and low-salt diets, serving as normotensive control animals for RRM-hypertensive rats, had dilator responses that were similar to those of rats that were on the high- and low-salt diets, but not subjected to the sham surgery. All rats were allowed to drink tap water, ad libitum. Body weights for normotensive rats maintained on either the low- or the high-salt diet
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were not different, while body weight in the RRMhypertensive group was significantly less than that of the normotensive controls on the low- or high-salt diet (Table 1). Methods and protocols for microcirculatory studies. On the day of the experiment, individual rats were anesthetized with an injection (60 mg z kg 21, ip) of pentobarbital sodium (Veterinary Laboratories, Lenexa, KS), and the trachea was cannulated to ensure a patent airway. The carotid artery and external jugular vein were cannulated for arterial pressure recording and for 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 (PSS), 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 2PO 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). The vessel selection procedure for these experiments was as follows: In a clearly visible region of the cremaster muscle, a second-order arteriole of approximately 60-mm diameter was identified. This vessel was tracked along its length, and successive branches were identified up to the point at which capillaries arose from the terminal arterioles. Once the capillaries arising from this arteriolar network were identified, the section of the arteriole that was immediately proximal to the capillaries was selected for analysis. In this selection procedure, the location of the arterioles (immediately proximal to the capillaries) was the selection criterion, and the absolute diameter of the arteriole was not a factor. At this point, the ultimate selection of the arterioles was subject to the following additional requirements: (1) location in a region of the muscle that was away from any incision, (2) clearly discernible vessel walls, (3) brisk flow velocity, and (4) active tone, as indicated by the occurrence of a brisk dilation in response to topical application of 10 24 M adenosine.
Arteriolar diameters were measured before and after the topical application of each of the following agents to the preparation: (1) acetylcholine (10 28–10 26 M), (2) iloprost (10 215–10 29 g z ml 21), (3) cholera toxin (10 29 g z ml 21), (4) sodium nitroprusside (10 28–10 26 M), and (5) forskolin (10 213–10 27 M). The maximum arteriolar diameter was also assessed in all arterioles by measuring the vessel response to superfusion with calcium-free physiological salt solution containing 10 24 M adenosine. Arteriolar diameters were measured after the vasodilator response to the individual agonists had stabilized, which occurred within 20 s of the agonist challenge. Successive agonist challenges were applied only after the vessel had returned to its resting diameter following the application of the preceding agonist. The application of the agents was randomized to prevent the possible occurrence of ordering effects and to control for any time-dependent changes in vascular reactivity during the experiment. The total experiment duration was approximately 150 min, and viability of the preparation was assessed by verifying that the vessels maintained normal levels of resting tone throughout the experiment. Data and statistical analysis. All data are presented as the mean 6 SEM of either the absolute vessel diameter (mm) or the change in diameter from rest in response to the agonist challenge (%). Analysis of variance was employed to determine initial differences across experimental conditions and Scheffe’s post hoc test was employed to identify significant differences between specific experimental groups. In all cases, a probability level of P , 0.05 was considered statistically significant.
RESULTS Mean arterial pressure and resting arteriolar diameter. Mean arterial pressure (MAP) and resting diameter of the cremasteric arterioles in the three experimental groups are summarized in Table 1. MAP was significantly elevated in RRM-hypertensive rats compared to either normotensive group. MAP in normotensive animals on the high-salt and low-salt diet was
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TABLE 1 Experimental Groups for Arteriolar Dilator Reactivity Studies Group
n
Age (weeks)
Mass (g)
MAP (mm Hg)
Diet
Normotensive Normotensive Hypertensive
12 12 12
12–13 12–13 12–13
356 6 18 362 6 19 318 6 12*†
119 6 4.6 123 6 3.4 139 6 3.3*†
LS HS HS
Rest diameter (mm) 18.0 6 1.0 18.8 6 1.0 19.3 6 0.9
Note. LS represents low-salt-content rat chow (0.4% NaCl), HS represents high-salt-content rat chow (4.0% NaCl). Asterisks represent significant differences between the age-matched groups compared to the normotensive controls on the low-salt diet. Daggers represent significant differences from normotensive controls on the high-salt diet.
not different. Resting diameter of the arterioles, prior to agonist application, was not significantly different across the three experimental groups. Responses to acetylcholine. Figure 1 summarizes the arteriolar responses to acetylcholine across the experimental groups. Arterioles of rats on the low-salt diet exhibited a significantly greater dilation than those of the normotensive controls on the high-salt
diet or the RRM-hypertensive rats. The dilation of arterioles in response to the higher concentrations of acetylcholine was also significantly greater in normotensive rats on the high-salt diet than in the RRMhypertensive animals. Responses to iloprost. Arteriolar responses to iloprost are summarized in Fig. 2. There were no significant differences in the response of arterioles to ilo-
FIG. 1. Responses of cremasteric arterioles to increasing concentrations of acetylcholine in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a LS diet; daggers indicate a significant reduction in the dilation compared to normotensive controls on a HS diet.
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FIG. 2. Responses of cremasteric arterioles to increasing concentrations of iloprost in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a LS diet; daggers indicate a significant reduction in the dilation compared to normotensive controls on a HS diet.
prost in the normotensive rats on low- and high-salt diets, except for the dilation in response to 10 29 g z ml 21, in which the arterioles of normotensive rats on the high-salt diet dilated significantly less than those of normotensive controls on a low-salt diet. In the RRM-hypertensive rats, arteriolar dilation in response to iloprost was significantly reduced relative to normotensive controls on either the low- or the highsalt diet. Responses to cholera toxin. In the animals on the low-salt diet, challenge with cholera toxin (10 29 g z ml 21) resulted in a 28 6 4.5% increase in the diameter of the cremasteric arterioles above their resting value (Fig. 3). This response was significantly larger than that of either the normotensive rats fed the highsalt diet (18 6 2.7%) or the RRM-hypertensive rats (12 6 1.3%). Responses to forskolin. Figure 4 summarizes the response of cremasteric arterioles to the topical appli-
cation of forskolin in the three experimental groups. There were no significant differences in the response of the arterioles to forskolin in normotensive animals on low-salt and high-salt diets. However, arteriolar responses to forskolin were significantly reduced in RRM-hypertensive animals compared to those in normotensive controls on the low- and high-salt diets. Responses to sodium nitroprusside. Arteriolar responses to sodium nitroprusside are summarized in Fig. 5. The vessel responses to nitroprusside were not significantly different in normotensive animals on low- and high-salt diets. However, arteriolar reactivity to nitroprusside was significantly decreased in the RRM-hypertensive rats compared to the normotensive controls on either the low-salt or the high-salt diet. Maximal dilation in response to calcium-free superfusate plus adenosine. Figure 6 summarizes the response of the cremasteric arterioles to superfusion with calcium-free PSS containing 10 24 M adenosine in
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FIG. 3. Responses of cremasteric arterioles to cholera toxin (10 29 g z ml 21) in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a low-salt diet.
LS, HS, and HSRRM rats. In these experiments, the maximum dilation from rest was not different in the normotensive rats on low-salt and high-salt diets, but was significantly reduced in the RRM-hypertensive rats.
DISCUSSION Recent work in our laboratory has demonstrated that a chronic elevation in dietary salt intake in normotensive rats and in RRM-hypertensive rats impairs the reactivity of skeletal muscle resistance arteries and distal arterioles to dilator agonists compared to responses in vessels from normotensive rats on low-salt
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diets. These altered reactivity patterns have been identified using an array of dilator agonists, each exerting its effect at either the endothelium or the vascular smooth muscle, and at different sites in the signal transduction pathway (Liu et al., 1997; Frisbee and Lombard, 1998). Preliminary investigations of the time course of the altered reactivity in skeletal muscle resistance arteries of normotensive rats during elevated dietary salt intake demonstrated that vessel responses to acetylcholine and iloprost are reduced compared to those of rats on a low-salt diet after as little as 3 days on the high-salt diet (Weber et al., 1997). In addition, Hansen-Smith et al. (1996) demonstrated that structural alterations in microvessels can occur over this same 3-day period in both RRM-hypertensive rats and normotensive animals on a high-salt diet. Taken to-
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FIG. 4. Responses of cremasteric arterioles to increasing concentrations of forskolin in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a LS diet; daggers indicate a significant reduction in the dilation compared to normotensive controls on a HS diet.
gether, these observations indicate that vascular adaptations to elevated salt intake and RRM hypertension may occur very rapidly. The present study builds on these observations to determine if the relaxation of cremasteric arterioles in response to endothelium-dependent and -independent dilator agonists is altered over a 3-day period in RRM-hypertensive rats and in normotensive rats on a high-salt diet, relative to normotensive controls on a low-salt diet. A further goal of these experiments was to determine whether structural remodeling of the arterioles, which would limit the ability of the vessels to increase their diameter in response to dilator stimuli, could contribute to an impaired relaxation of arterioles in response to vasodilator agonists in RRMhypertensive rats or in normotensive animals subjected to short-term increases in dietary salt intake. Responses to vasodilator agonists. Interpretation of the patterns of arteriolar reactivity in response to the agonist challenges in this study indicated a clear
trend for a decreased responsiveness to the endothelium-dependent agonist acetylcholine, the smooth muscle receptor-dependent agonist iloprost, and the heterotrimeric G-protein activator cholera toxin in normotensive rats on the high-salt diet and in RRMhypertensive rats, compared to the normotensive controls on a low-salt diet. While similar patterns of impaired vasodilator reactivity in cremasteric arterioles have been previously identified over a chronic (4week) time frame in RRM-hypertensive rats and in normotensive control animals on a high-salt diet (Frisbee and Lombard, 1998), the present study presents the first observations demonstrating conclusively that the impaired vascular reactivity in RRM-hypertensive rats and normotensive rats on a high-salt diet develops in as little as 3 days. A crucial question regarding the impaired vasodilator responses in the hypertensive rats and in the normotensive rats on high-salt diet is the mechanism of the impaired relaxation. Specifically, is the reduced
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FIG. 5. Responses of cremasteric arterioles to increasing concentrations of sodium nitroprusside in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a low-salt diet.
dilator response due to an impaired function of the mechanisms mediating active relaxation of the vascular smooth muscle, to structural alterations in the arteriolar wall that impair the ability of the vessel to dilate, or to both? This question is of added interest, given the very short time period (3 days) over which impaired vasodilator responses develop, which is much more rapid than previously established. If the reduced dilation of arterioles in response to the different vasodilator agents in RRM-hypertensive rats and in normotensive controls on a high-salt diet is due to alterations in active vasodilator mechanisms, this could have resulted from compromised function at several sites in the transduction pathway. This conclusion is based on the observation that arteriolar relaxation was impaired during the application of several dilator agents with different mechanisms of action. These include acetylcholine, which acts on receptors on the vascular endothelium; iloprost, which
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acts directly on receptors on the vascular smooth muscle cell membrane; and cholera toxin, which activates the G S protein leading to relaxation of cremasteric arterioles (Stekiel et al., 1993). Alternatively, this general impairment of active vessel relaxation in response to a wide variety of vasodilator stimuli may reflect a defect at a common point in the signal transduction pathway that is downstream of the site of action of the various vasodilator agonists, e.g., defective K 1 channel regulation, defective regulation of intracellular Ca 21 levels, etc. The hypothesis that the mechanisms responsible for active vasodilation are impaired in arterioles of animals with RRM hypertension is consistent with the results of Stekiel et al. (1993), who reported that stimulation of b-adrenergic receptors of first-order arterioles with isoproterenol and activation of G proteins with cholera toxin had no effect on the transmembrane potential of arteriolar smooth muscle cells in RRM-
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FIG. 6. Maximal dilation of cremasteric arterioles in response to superfusion with Ca 21-free PSS containing 10 24 M adenosine in normotensive animals fed low-salt (LS) or high-salt (HS) diets and in reduced renal mass hypertensive rats fed a HS diet (HSRRM). Data are expressed as mean (6SE) percentages increase from the rest diameter, measured during superfusion with PSS for 12 HSRRM rats, 12 HS rats, and 12 LS rats. Asterisks indicate a significant reduction in the dilation compared to normotensive controls on a low-salt diet.
hypertensive rats, but hyperpolarized the vascular smooth muscle in normotensive controls. Those observations indicated that the b-receptor-mediated cascade leading to smooth muscle hyperpolarization and relaxation was depressed with chronic RRM hypertension, and suggested that the specific site of this depression may have been at the G S protein–adenylyl cyclase coupling stage. In addition, preliminary studies by our group indicate that a short-term (3 days) elevation of dietary salt intake eliminates vascular smooth muscle hyperpolarization and vascular relaxation in middle cerebral arteries of normotensive rats (Lombard et al., 1997), which is also consistent with the interpretation that some of the mechanisms mediating active relaxation of the vessels in response to different vasodilator stimuli are impaired by exposure to a high-salt diet.
Structural remodeling in arterioles of rats on a highsalt diet. An alternative interpretation of the results of the present experiments is that the diminished relaxation of vessels of the RRM-hypertensive rats and the normotensive rats on the high-salt diet in response to the agonist challenges is due to structural alterations in the arteriolar wall of these animals, which could physically hinder the ability of the vessels to increase their diameter, even in the absence of defects in active vasodilator mechanisms. The interpretation that structural alterations can occur rapidly in rats with acute RRM hypertension is consistent with reports in the literature (Rorive et al., 1980; Loeb et al., 1986; Hansen-Smith et al., 1996) and with our present observation of a significant reduction in the response of cremasteric arterioles to superfusion with Ca 21-free PSS plus adenosine in hypertensive animals. This re-
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duced response to Ca 21-free PSS plus adenosine may be indicative of the earliest stages of structural remodeling of the arterioles in animals with RRM hypertension. In contrast, there was no evidence in the present study for structural remodeling of arterioles in response to acute elevations in dietary salt intake alone. However, in our earlier study, vascular remodeling was identified in normotensive rats after a chronic elevation of dietary salt intake (Frisbee and Lombard, 1998). Taken together, these observations suggest that structural changes in normotensive rats on a high-salt diet develop more slowly in the absence of the elevated blood pressure that is associated with the development of hypertension. However, the relative magnitude of the relaxation of the arterioles in response to the various vasodilator agonists used in the present study suggests that it is unlikely that structural remodeling was the sole factor leading to the impaired relaxation of the vessels of the hypertensive rats and normotensive animals on the high-salt diet. In these experiments, the magnitudes of the maximal dilation of the cremasteric arterioles during superfusion with Ca 21-free PSS containing 10 24 M adenosine in normotensive rats on the high-salt diet and in RRM-hypertensive rats (71.8 6 12.1 and 59.1 6 6.5% above the initial rest vessel diameter, respectively) were much greater than the increase in vessel diameter in response to any of the dilator agonists in the present study. This observation suggests that an inability of the vessel to increase its diameter due to structural remodeling was not the only factor responsible for the reduced dilation in response to the agonists used in this study. However, it is conceivable that structural alterations could reduce the distensibility of arterioles in animals with acute RRM hypertension and in animals subjected to short-term elevations in dietary salt intake, thereby interfering with the ability of the vessels to increase their diameter following relaxation of the vascular smooth muscle. Potential mechanisms for the altered microvessel structure and function. Previous studies have suggested that the renin–angiotensin system may play a critical role in the maintenance of the normal structure of the microcirculation and that this protective effect may be lost when circulating angiotensin II is suppressed during RRM hypertension and elevated di-
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Frisbee and Lombard
etary salt intake (Hernandez et al., 1992; Rieder et al., 1997). Preliminary investigations in our laboratory have suggested that the suppression of the renin– angiotensin system with high-salt diet may also play a critical role in contributing to the impaired vascular reactivity in rat skeletal muscle resistance arteries (Weber et al., 1997) and in rat middle cerebral arteries (Lombard et al., 1997). In those studies, chronic intravenous infusion of a low dose of angiotensin II in rats maintained on a high-salt diet restored the normal vasodilator reactivity that was suppressed in arteries of animals on a high-salt diet that did not receive angiotensin II infusion. Taken together, these experiments suggest that angiotensin II suppression may be a critical element regulating the alterations in microvessel structure and function that develop with RRM hypertension and high-salt diet. Conclusions. When considered in combination with our existing work (Frisbee and Lombard, 1998; Weber et al., 1997; Liu et al., 1997; Lombard et al., 1997) and the findings of other studies using the one-kidney, one-clip model of hypertension (Hashimoto et al., 1987), the observations reported in the present study can begin to be integrated into a conceptual model addressing microvascular adaptations to the development of RRM hypertension and elevations in salt intake. It is evident that, with the development of hypertension, the ability of skeletal muscle resistance arteries and arterioles to relax in response to vasodilator stimuli is rapidly impaired. Structural alterations appear to develop at a slower rate and occur earlier in hypertensive animals than in normotensive controls. As the duration of the RRM hypertension and the period of elevated salt intake is increased, the remodeling becomes more extensive, and the anatomic adaptations become clearly evident both during chronic elevations of salt intake in normotensive animals and in RRM hypertension. The mechanisms and interplay between these physiological and anatomic processes, the potential for reversibility of the changes in structure and reactivity, the role of the renin–angiotensin system in contributing to these changes, and the functional implications of these physiological and anatomical changes remain to be identified and represent exciting areas for future investigation.
Microvessels, Salt Intake, and Hypertension
ACKNOWLEDGMENTS This work was supported by NIH Grants HL29587, HL37374, and HL52211. The present studies were reported in preliminary form at the 1998 Microcirculatory Society meetings in San Francisco, California. The authors thank the Berlex Corporation for their generous donation of the iloprost used in these experiments.
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Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.