Enhanced angiotensin-converting enzyme activity and systemic reactivity to angiotensin II in normotensive rats exposed to a high-sodium diet

Abstract

A high salt diet is associated with reduced activity of the renin–angiotensin–aldosterone system (RAAS). However, normotensive rats exposed to high sodium do not show changes in systemic arterial pressure. We hypothesized that, despite the reduced circulating amounts of angiotensin II induced by a high salt diet, the cardiovascular system’s reactivity to angiotensin II is increased in vivo, contributing to maintain arterial pressure at normal levels. Male Wistar rats received chow containing 0.27% (control), 2%, 4%, or 8% NaCl for six weeks. The high-sodium diet did not lead to changes in arterial pressure, although plasma levels of angiotensin II and aldosterone were reduced in the 4% and 8% NaCl groups. The 4% and 8% NaCl groups showed enhanced pressor responses to angiotensin I and II, accompanied by unchanged and increased angiotensin-converting enzyme activity, respectively. The 4% NaCl group showed increased expression of angiotensin II type 1 receptors and reduced expression of angiotensin II type 2 receptors in the aorta. In addition, the hypotensive effect of losartan was reduced in both 4% and 8% NaCl groups. In conclusion these results explain, at least in part, why the systemic arterial pressure is maintained at normal levels in non-salt sensitive and healthy rats exposed to a high salt diet, when the functionality of RAAS appears to be blunted, as well as suggest that angiotensin II has a crucial role in the vascular dysfunction associated with high salt intake, even in the absence of hypertension.

1. Introduction

The effects of sodium intake on the functionality of the renin–angiotensin–aldosterone system (RAAS) have been widely investigated in the past decades, revealing that sodium restriction in healthy animals results in increased angiotensin II levels in plasma, accompanied by reduced vascular reactivity, while excessive ingestion of sodium leads to decreased production of angiotensin II but augmented stimulatory effects of this mediator in vitro [2,6,42]. Despite the recent studies addressing this field, the physiology, functionality and importance of the RAAS for the effects of high salt intake on the cardiovascular system of healthy subjects remain poorly understood, mainly because the vast majority of these studies were performed in Dahl salt-sensitive, corticosterone acetate (DOCA)-salt or spontaneously hypertensive rats. In these animal models, high salt intake has been often associated with reduced activity or circulating levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II, or aldosterone [4,16,24]. Nevertheless, these same studies revealed that the high salt diet increased angiotensin II and aldosterone in kidney and heart of Dahl salt-sensitive [4], as well as the plasma levels of angiotensinogen and angiotensin II in spontaneously hypertensive rats [16]. Interestingly, Kobori and co-workers found that exposure to a high salt diet reduced plasma renin and angiotensinogen in both Dahl salt-sensitive and Dahl salt-resistant rats, but only Dahl salt-sensitive rats presented increased levels of angiotensinogen in kidneys and urine [24]. All together, these studies suggest that in salt-sensitive and spontaneously hypertensive rats, the excessive ingestion of salt may result in increased production or activation of components of RAAS in tissues, such as kidneys, in spite of their reduced levels in plasma. Indeed, it suggests that angiotensin II plays a role in the vascular dysfunction associated with high salt ingestion in experimental models of hypertension. However, the effects of high salt intake in the in vivo functionality of RAAS in non-hypertensive and healthy animals remain unclear.

Using molecular approaches, it was demonstrated that high sodium intake increases both the mRNA levels and protein expression of angiotensin II type 1 (AT1) receptors in the aorta and vascular smooth muscle cells [30], and reduces the expression of angiotensin II type 2 (AT2) receptors in small mesenteric arteries [13] of normotensive Sprague–Dawley rats. However, none of the previous studies investigated the impact of high salt-induced changes in the RAAS on the systemic pressor effects of angiotensin II. We hypothesized that, in spite of the reduced circulating amounts of angiotensin II induced by a high salt diet, the cardiovascular system becomes more responsive to the renin–angiotensin system, improving its ability to convert angiotensin I to angiotensin II and increasing the reactivity to angiotensin II in vivo. This modulation at multiple levels could explain why the arterial pressure of healthy animals exposed to excessive amounts of salt is not impaired, even when the total circulating amounts of angiotensin II in plasma are greatly reduced.

2. Material and methods

2.1. Animals and experimental protocols

All procedures adopted in this study were approved by the Institutional Ethics Committee for Animal Use of the Universidade Federal do Paraná (authorization number 345), and by the Institutional Animal Care and Use Committee of Georgia Health Sciences University (authorization number 2011-0353). We used male Wistar rats provided by either Universidade Federal do Paraná (Curitiba, PR, Brazil) or Harlan Laboratories (Indianapolis, IN, USA). The animals were kept under standard laboratory conditions, with a constant 12-hour light/dark cycle and controlled temperature (22 ± 2 °C). Both water and food were supplied ad libitum. The study included four distinct experimental groups. The first group, used as control, received standard rat chow (purchased from Nuvital®, Curitiba, PR, Brazil or Teklad Global Diets®, Indianapolis, IN, USA), containing the regular amount of NaCl (0.27%). The other three groups received rat chow containing 2%, 4%, or 8% NaCl. This exposure to high amounts of NaCl was started at weaning and continued for six weeks, when the experiments were performed.

2.2. Evaluation of cardiovascular reactivity in anesthetized rats

At the end of the sixth week, the rats were anesthetized by intramuscular injection of ketamine and xylazine (100/20 mg/kg), and surgically prepared for direct blood pressure measurement, as previously described [9]. The mean arterial pressure (MAP) was assessed through a catheter inserted into the carotid artery, and connected to a pressure transducer coupled to a digital recording system (MacLab®) and its software (Chart, v 4.0), both from AD Instruments (Castle Hill, Australia). After the surgical process, there was an interval of 20 min to allow for blood pressure stabilization before the injection of any drug.

After stabilization, different animals received intravenous injections containing either angiotensin I (3, 10 and 30 pmol/kg), angiotensin II (3, 10 and 30 pmol/kg), or bradykinin (3, 10 and 30 nmol/kg). The doses were injected in a total volume of 250 μL (including washing of the catheter). An interval of 10 min was allowed for MAP stabilization between each administration. The maximal effect on MAP (in mm Hg) and the duration of responses (in s) were calculated and compared among the groups.

In a separated series of experiments, the effects of angiotensin II on MAP were evaluated before and 5 min after a single administration of losartan (1 mg/kg, given as intravenous bolus injection), a selective angiotensin II AT1 receptor antagonist. The ability of losartan to reduce the pressor effects of angiotensin II in control and 4% NaCl groups was evaluated in this approach. In addition, the hypotensive effects of losartan (3, 10, and 30 mg/kg) were evaluated in all experimental groups.

The animals were killed with an overdose of thiopental (over 40 mg/kg, administered by intravenous injection). In some experiments, the thoracic aorta was removed and quickly frozen in liquid nitrogen for subsequent Western blot analysis.

2.3. Measurement of systolic blood pressure in conscious rats

To verify if the absence of hypertension in animals subjected to a high salt diet in our experiments could be related to the anesthetic drugs used, systolic blood pressure was measured in conscious rats at 2, 3, 4, 5 and 6 weeks of exposure to regular (control) or 4% NaCl chow. These experiments were performed using a non-invasive volume pressure recording sensor that was placed around the tail, allowing the measurement of arterial pressure parameters in a digital recording system (CODA System, Kent Scientific, Torrington, CT, USA).

2.4. Measurement of diuresis

In order to evaluate the influence of a high salt diet on diuresis and natriuresis, rats exposed to diets containing 2%, 4% and 8% NaCl (as well as animals belonging to the control group) were individually accommodated in metabolic cages for 8 h (with free access to water), allowing the continuous measurement of urinary output. To avoid the influence of hormones endogenously released at different times of the day, these experiments were always performed from 8 AM to 4 PM. In addition, the urine collected was checked for density, pH, and sodium concentration [22]. These experiments were performed once a week during the exposure to a high salt diet.

2.5. Biochemical assays

2.5.1. ACE assay

At the end of six weeks of receiving the high salt diet, blood samples were collected from animals that had not been subjected to pharmacological manipulation. The blood was centrifuged (800 g for 15 min) for plasma separation. The plasma samples were kept at −80 °C until the assays were performed. For the ACE assay, 10 μL of plasma was incubated with 490 μL of the assay solution (composition: Hip-His-Leu at 5 mM in 0.4 M sodium borate buffer, pH 8.3) for 15 min at 37 °C. The reaction was stopped by addition of 1.2 mL of NaOH (0.34 N). The product, His-Leu, was measured fluorometrically (365 nm excitation and 495 nm emission, Aminco Model J4-7461 fluoromonitor, American Instrument Co., Silver Spring, MD, USA) after the addition of 100 μL of o-phthaldialdehyde (20 mg/mL) in methanol for 10 min, followed by 200 μL of HCl (3 N) and centrifugation at 800 g for 5 min at room temperature [37]. To correct for the intrinsic fluorescence of plasma, time-zero blank samples were prepared by adding plasma after NaOH treatment. All measurements were made in triplicate.

2.5.2. Angiotensin II and aldosterone assays

For these experiments, blood samples from the control, 4% and 8% NaCl groups (without any pharmacological manipulation) were collected immediately after the induction of anesthesia by oxygen–isoflurane (3%) inhalation. The blood was put into glass tubes containing 7.5% ethylenediaminetetraacetic acid (EDTA), and centrifuged for plasma separation (800 g for 15 min). Angiotensin II levels in the plasma were measured by enzyme immunoassay immediately after methanol extraction, as previously described [40]. The concentration of aldosterone in the plasma was measured by enzyme-linked immunosorbent assay (ELISA; Immuno-Biological Laboratories, Inc., Minneapolis, MN, USA), according to the manufacturer’s instructions. All measurements were made in duplicate.

2.5.3. Detection of angiotensin II receptors by Western blotting

The expression levels of AT1 and AT2 receptors were evaluated in thoracic aortas obtained from the control and 4% NaCl groups. After removal from the animal, the entire thoracic aorta was quickly frozen in liquid nitrogen and maintained at −80 °C until it was processed for protein purification and subsequent electrophoretic separation, using 40 μg of protein per well in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) run in the Mini-PROTEAN® Tetra cell apparatus connected to a PowerPac™ HC power supply (both from Bio-Rad, CA, USA). Protein quantification and electrophoresis followed the same protocol and the same reagents were used as previously described [46]. The proteins were electrophoretically transferred to nitrocellulose membranes (Hybond; Amersham Biosciences, NJ, USA), and subjected to poly-clonal anti-AT1 and anti-AT2 receptors (Santa Cruz Biotechnology, Dallas, TX, USA), or monoclonal anti-actin (Sigma-Aldrich, St. Louis, MO, USA) primary antibodies overnight at 4 °C, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The membranes were washed and exposed to chemiluminescent substrate for HRP (Pierce Biotechnology, Rockford, IL, USA) for protein detection using a FluorChem® HD2 Imaging System (Alpha Innotech Corp., Santa Clara, CA, USA). The bands were quantified by densitometry using UN-SCAN-IT gel software (Silk Scientific, Inc., Orem, UT, USA).

2.6. Drugs and reagents

Angiotensin I, angiotensin II, bradykinin, o-phthaldialdehyde and hippuryl-L-histidyl-L-leucine were obtained from Sigma (St. Louis, MO, USA). Losartan was donated by Bula Verdde Pharmacy (Curitiba, PR, Brazil). The angiotensin II measurement kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Antibodies against angiotensin I and II receptors were produced by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bradykinin, angiotensin I, and angiotensin II (stock solutions) were dissolved in HCl (0.1 N), and diluted in regular isotonic saline solution (0.9% NaCl) immediately before each experiment.

2.7. Statistical analysis

The results are expressed as mean ± standard error of the mean (SEM) of five to eight experiments per group. The data were analyzed by one- or two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test, or Student’s t test, when applicable. A value of P < 0.05 was considered statistically significant. Graphs were drawn and statistical analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Enhanced responsiveness to angiotensin I and activity of ACE after high salt intake

Administration of angiotensin I led to dose-dependent pressor effects in the 2% NaCl group that did not differ from control, but significantly enhanced pressor effects were observed in the 4% and 8% NaCl groups (Fig. 1). For instance, with the intermediate dose of angiotensin I used in our experiments (10 pmol/kg), MAP was increased by 53.1 ± 5.8 and 55.5 ± 5.8 mm Hg in the 4% and 8% NaCl groups, respectively, and by 29.5 ± 2.8 mm Hg in the control group (P < 0.05, comparing the control group with the 4% and 8% NaCl groups).

Fig. 1.

Enhanced pressor effects of angiotensin I in normotensive rats exposed to a high-sodium diet for six weeks. Increase in mean arterial pressure (a, c and e) and duration of the pressor effect (b, d and f) of intravenously administered angiotensin I (3, 10 and 30 pmol/kg). Angiotensin I was administered in bolus in anesthetized rats previously subjected to high NaCl (2, 4 or 8%) chow. The results shown are mean ± SEM (n = 6–7). Statistical analyses were performed by two-way analysis of variance followed by Bonferroni’s post hoc test. *P < 0.05 compared with the respective control group.

Quantitative analysis of ACE activity revealed a significant increase (approximately 20%) in plasma samples obtained from the 8% NaCl group compared with control, but there was no increase in samples obtained from the 2% and 4% NaCl groups (Fig. 2a). This ex vivo activity was functionally confirmed in vivo by a reduced duration in the hypotensive effect of bradykinin in rats from the 8% NaCl group (Fig. 2b), with no change in the peak of hypotensive responses (data not shown). Animals from the 2% and 4% NaCl groups did not show impaired responses to bradykinin (data not shown).

Fig. 2.

Increased plasma angiotensin-converting enzyme (ACE) activity and reduced duration of the hypotensive effect of bradykinin in normotensive rats fed with 8% NaCl chow. Plasma ACE activity (a) was measured in samples collected from the 2%, 4% and 8% NaCl groups. The letter ‘C’ indicates the control group. The duration of the hypotensive effect of bradykinin after its intravenous administration in anesthetized rats (b). The results shown are mean ± SEM (n = 5–12). Statistical analyses were performed by one-way (a) or two-way analysis of variance (b), followed by Bonferroni’s post hoc test. *P < 0.05 compared with the respective control group.

3.2. Increased effects of angiotensin II after high salt intake

Although the rats exposed to chow containing 2% NaCl did not show altered responses to angiotensin II (Fig. 3a), increased reactivity to this peptide was found in animals from the 4% and 8% NaCl groups. For instance, the pressor effects of the intermediate dose of angiotensin II tested in our experiments (10 pmol/kg) were significantly increased from 39.5 ± 4.7 (control) to 54.6 ± 5.7 and 63.3 ± 6.0 mm Hg in the 4% and 8% NaCl groups, respectively (Fig. 3b and c). In addition, the duration of the effects of 3, 10 and 30 pmol/kg angiotensin II was extended, respectively, from 94.9 ± 17.2, 152.2 ± 19.1, and 189.5 ± 22.0 s in the control group, to 451.1 ± 82.8, 579.5 ± 55.2, and 628.9 ± 114.7 s in the 8% NaCl group (P < 0.05, for all doses tested).

Fig. 3.

Augmented pressor responses to angiotensin II in normotensive rats exposed to a high-sodium diet for six weeks. Increase in mean arterial pressure induced by angiotensin II (administered in bolus) in rats from the 2% (a), 4% (b), and 8% (c) NaCl groups. The effects were measured in anesthetized rats after six weeks of exposure to a high-sodium diet. The results shown are mean ± SEM (n = 5–6). Statistical analyses were performed by two-way analysis of variance followed by Bonferroni’s post hoc test. *P < 0.05 compared with the control group.

3.3. Unaltered arterial pressure in normotensive rats exposed to a high salt diet

In spite of the observed enhanced effects of angiotensin I and II, animals subjected to 2%, 4% or 8% NaCl from weaning for six weeks did not show any change in heart rate or arterial pressure, as measured in anesthetized rats before the administration of vasoactive drugs (Table 1). Similarly, systolic arterial pressure measured in conscious animals did not differ between the 4% NaCl and control groups (Fig. 4).

Table 1.

Arterial pressure and heart rate of anesthetized rats exposed to high sodium intake for six weeks from weaning.

Group	SAP (mm Hg)	DAP (mm Hg)	MAP (mm Hg)	HR (bpma)
Control	101.3 ± 4.0	68.4 ± 4.8	82.4 ± 4.2	227.1 ± 21.7
2% NaCl	94.8 ± 3.9	58.5 ± 3.9	73.6 ± 3.5	203.7 ± 15.5
4% NaCl	101.9 ± 1.9	63.2 ± 3.5	78.3 ± 2.6	220.4 ± 10.7
8% NaCl	107.2 ± 3.4	63.5 ± 5.0	80.5 ± 4.6	202.0 ± 18.3

The results shown are mean ± SEM (n = 6–7). Systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), and heart rate (HR). No statistical differences were found.

^abpm: Beats per minute.

Fig. 4.

Systolic arterial pressure of conscious rats exposed to high-sodium intake for six weeks. The results shown are mean ± SEM (n = 16). Statistical analyses were performed by two-way analysis of variance followed by Bonferroni’s post hoc test. No statistically significant differences were found.

3.4. Reduced levels of circulating angiotensin II and aldosterone and increased diuresis after high salt intake

Levels of angiotensin II and aldosterone were reduced in the plasma samples of animals exposed to chow containing 4% and 8% NaCl. Plasma levels of angiotensin II (89.3 ± 21.4 pg/mL in the control group) were reduced by 70 to 80% in the 4% and 8% NaCl groups (Fig. 5a). Likewise, aldosterone levels (16.4 ± 4.0 ng/mL in the control group) were reduced by 80 and 60% in the 4% and 8% NaCl groups, respectively (Fig. 5b). In accordance with the reduced plasma levels of aldosterone, rats exposed to a high salt diet (the 4% and 8% NaCl groups) showed a significant increase in urinary output (30 to 150%) in all weeks of treatment (Fig. 6a). Similarly, urinary excretion of sodium was significantly increased (Fig. 6b). The density and pH of urine were unchanged at all analyzed time points (data not shown).

Fig. 5.

Reduced plasma levels of angiotensin II and aldosterone in normotensive rats exposed to a high-sodium diet for six weeks. Angiotensin II (a) and aldosterone (b) were measured in plasma collected from rats exposed to regular chow (control) or high NaCl (4 and 8%) chow. The results shown are mean ± SEM (n = 5–7). Statistical analyses were performed by one-way analysis of variance followed by Bonferroni’s post hoc test. *P < 0.05 compared with the control group.

Fig. 6.

Increased diuresis and natriuresis in normotensive rats exposed to a high-sodium diet for six weeks. Urinary volume (a) and amount of sodium excreted (b) by rats exposed to control or high salt diet for six weeks. The results shown are mean ± SEM (n = 5–8). Statistical analyses were performed by two-way analysis of variance followed by Bonferroni’s post hoc test. *P < 0.05 compared with the control group.

3.5. Altered expression of AT1 and AT2 receptors and effects of losartan after high salt intake

As revealed by Western blot analyses, the aorta of rats exposed to chow containing 4% NaCl showed increased expression of AT1 receptors compared with control (Fig. 7a). On the other hand, the expression of AT2 receptors was reduced in the 4% NaCl group (Fig. 7b).

Fig. 7.

Impaired expression and functionality of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the aorta of normotensive rats exposed to a high-sodium diet for six weeks. Protein expression of AT1 (a) and AT2 (b) receptors in the aorta of the control and 4% NaCl groups was measured by Western blotting. The values were normalized by β-actin expression and shown in arbitrary units. Reduced inhibition of the pressor effects of angiotensin II (c) and diminished hypotensive effects (d) induced by losartan in NaCl groups. The results shown are mean ± SEM of five samples, obtained from different rats (a and b), or five-six animals per group (c). Statistical analyses were performed by Student’s t test (a and b) or two-way analysis of variance followed by Bonferroni’s post hoc test (c and d). *P < 0.05 compared with the control group.

Administration of AT1 receptor antagonist losartan (1 mg/kg, i.v.) significantly reduced the effects of all doses of angiotensin II in control animals (Fig. 7c, black bars), but it failed to inhibit the pressor effects induced by the higher doses of angiotensin II (10 and 30 pmol/kg) in the 4% NaCl group (Fig. 7c, white bars). Importantly, this small dose of losartan did not change the basal systemic arterial pressure in any of our experimental groups. However, the intravenous injection of 3, 10 and 30 mg/kg of losartan reduced the MAP of control animals by 9.6 ± 1.3, 12.5 ± 1.1 and 21.3 ± 2.6 mm Hg, respectively. Interestingly, both 3 mg/kg (Fig. 7d) and 10 mg/kg (data not show) presented reduced effects in animals from 4% and 8% NaCl groups. However, the hypotensive effect obtained after administration of losartan at 30 mg/kg did not differ between the control and NaCl groups (21.3 ± 2.6, 27.9 ± 4.8, 29.1 ± 5.3 and 11.9 ± 6.6 mm Hg, for control and 2%, 4%, and 8% NaCl groups, respectively).

4. Discussion

In this study, using normotensive and healthy Wistar rats, we found that animals fed chow containing 4% and 8% NaCl displayed augmented pressor responses to intravenously administered angiotensin I and angiotensin II. Both the peak of the responses and the duration of the effects were significantly increased. Studies, performed mainly in salt-sensitive and hypertensive rats, have suggested that RAAS is inhibited by high salt intake, as evidenced by reduced activity or levels of renin, angiotensinogen, ACE, angiotensin II and aldosterone in the plasma of these animals [4,16,24,33], although augmented tissue levels of angiotensinogen, angiotensin II and aldosterone have been found mainly in kidneys [4,24]. These studies indicate that local activation of components of RAAS may be involved in the vascular dysfunction and maintenance of hypertension in experimental models of hypertension associated with a high salt diet, in spite of the reduced systemic activation of RAAS. However, the in vivo functionality of RAAS in normotensive animals fed a high salt diet remains poorly understood. Indeed, at least to our knowledge, this is the first study using in vivo approaches to demonstrate that in spite of the reduced levels of circulating angiotensin II, animals subjected to a high salt diet present increased pressor responses to angiotensin II and reduced hypotensive effects mediated by losartan, both associated with augmented expression of angiotensin II AT1 receptors.

Using blood samples collected from anesthetized rats, a strategy that may have increased the levels of angiotensin II found in the plasma of high salt animals (as well as in control group) [7,19], we did confirm that both angiotensin II and aldosterone were reduced in the plasma of Wistar rats after six weeks under high salt intake (Fig. 5). Nevertheless, our study also demonstrates that in spite of these findings, the capacity of angiotensin II to increase systemic arterial pressure is maintained or even increased in healthy rats subjected to a high salt diet.

The enhanced reactivity to angiotensin II has been described in several in vitro studies and has been associated with augmented expression of AT1 receptors [30] or reduced expression of AT2 receptors [2,34,36], which mediate angiotensin II-induced vasodilation [41]. For comparative purposes, we also evaluated the expression levels of both AT1 and AT2 receptors in vessels removed from rats subjected to high salt intake. The results confirmed the findings of previous studies, demonstrating increased expression of AT1 receptors and a simultaneous reduction in the levels of AT2 receptors (Fig. 7). In order to better explore the role of augmented expression of AT1 receptors in the increased reactivity to angiotensin II found after the high-sodium diet in our in vivo experiments, we evaluated the effects of a single dose of losartan, a selective AT1 receptor antagonist, against the pressor effect of angiotensin II in both control and 4% NaCl group. Losartan similarly inhibited the effects of the smallest dose of angiotensin II in both control and 4% NaCl groups, but failed to reduce the effects of the highest doses of angiotensin II in high salt animals (Fig. 7c). We also verified the ability of losartan to induce hypotension in both control and NaCl groups. For this purpose, the animals received bolus intravenous injection of losartan at 3, 10 or 30 mg/kg. Interestingly, the smallest dose of losartan was able to induce a transitory hypotensive effect in control and 2% NaCl groups, which was almost abolished in 4% and 8% NaCl groups (Fig. 7d). A similar profile of effect was observed even when the intermediary dose (10 mg/kg) of losartan was tested (data not shown). On the other hand, the highest dose (30 mg/kg) was able to induce hypotension in both control and NaCl groups (see Results section for details), indicating that although less sensitive to losartan, the AT1 receptors present in the vascular system of rats fed a high salt diet remain fully functional and under influence of endogenous angiotensin II, in spite of the reduced plasma levels of angiotensin II found in these animals. Taken together, these data suggest that the enhanced effects of angiotensin II found in vivo after high salt intake are, at least in part, a consequence of the up-regulation of AT1 receptors, likely down-regulation of AT2 receptors in the vascular system.

Interestingly, animals subjected to a high salt diet showed a more pronounced hyperresponsiveness to angiotensin I compared with angiotensin II (Figs. 1 and 3). Angiotensin I is converted to angiotensin II by ACE. Indeed, the pressor effects observed after the administration of angiotensin I are induced by angiotensin II. Therefore, we investigated if the augmented reactivity to angiotensin I in animals from the high salt groups might involve changes in the activity of ACE. The results revealed that the activity of ACE was significantly increased in the plasma of animals subjected to 8% NaCl in their chow, while it was not impaired in animals fed with 2 and 4% NaCl chow (Fig. 2a). The activity of ACE has been widely investigated in animals subjected to different levels of salt intake and is generally acceptable that low sodium consumption can up regulate its activity [12]. On the other hand, using Wistar-Kyoto rats fed with chow containing 8% NaCl for 21 days Ingert and co-workers found a similar increase in both plasma and renal ACE activity, accompanied by increased angiotensinogen levels and reduced renin activity [20]. Although we have not been able to measure the plasma levels of angiotensinogen and renin in our experiments, a reduction in plasma renin activity after excessive sodium intake has been found by other previous studies [23]. Taken together with our data, these studies suggest that the reduced levels of angiotensin II found in the plasma of healthy rats exposed to a high salt diet result from a decreased availability of angiotensin I, rather than from a deficit in the ability of ACE to catalyze the conversion of angiotensin I to angiotensin II, since plasma ACE activity is maintained or increased in these animals (Fig. 2). Importantly, our study suggests that in animals subjected to very high amounts of sodium (8% NaCl group) during six weeks, the increased activity of ACE contributes to avoid an even greater reduction in plasma levels of angiotensin II.

We also demonstrated that the duration of the hypotensive effect of bradykinin, an endogenous vasodilator that is inactivated by ACE, was significantly reduced in rats from the 8% NaCl group (Fig. 2b), confirming the biological relevance of the augmented plasma ACE activity found in this group. Bradykinin, acting as an endogenous agonist of constitutive bradykinin B2 receptors in endothelial cells, is well known by its ability to stimulate the production of endothelium-derived relaxing factors [38], such as nitric oxide [5,14], prostacyclin [43], and endothelium-derived hyperpolarizing factor [11,28]. Moreover, activation of bradykinin B2 receptors have been implicated in the production of nitric oxide mediated by stimulation of AT2 receptors in different tissues, including kidneys [1] and uterine artery [15], among others [3]. Thus, the increased activity of ACE explains the reduced duration of the hypotension induced by exogenous bradykinin in 8% NaCl groups. In addition, taken into account the physiological roles of bradykinin in the vascular system, this finding suggests that a decreased bioavailability of endogenous bradykinin mediated by ACE may contribute to reduce the release of endothelium-derived relaxing factors in animals subjected to the high salt diet, which in turn may explain, at least in part, the increased duration of the pressor effect of angiotensin II found in our study.

Epidemiological studies have shown a correlation between excessive dietary salt intake and increased prevalence and incidence of essential hypertension [10]. Nonetheless, after being on a high salt diet for six weeks, none of the animals in the current study showed hypertension, even when arterial pressure was assessed in conscious animals (Fig. 4). Unlike measurements performed in anesthetized rats, assessment of arterial pressure in conscious animals retains all the effects of salt on the central nervous system, including the previously described sympathoexcitatory action associated with salt-induced hypertension in salt-sensitive subjects [8,17,18]. Interestingly, high salt-induced hypertension can be easily achieved by combining sodium overload with mineralocorticoid stimulation (by deoxycorticosterone acetate) [21,39], or angiotensin II administration [26], but it is rarely described in normotensive rats. Our experiments were performed in healthy rats (not salt-sensitive, previously hypertensive, or treated with mineralocorticoids or angiotensin II). The exposure of these animals to a high salt diet resulted in an intense natriuresis accompanied by elevated urine flow (Fig. 6), with no change in plasma sodium (data not shown), which may explain the lack of hypertension in healthy rats, as previously demonstrated [29]. Importantly, even in the absence of hypertension, excessive consumption of sodium has been associated with deleterious effects on the cardiovascular system, such as fibrosis in the left ventricle and intramyocardial vessels, and left ventricular and renal hypertrophy [44,45]. Interestingly, in normotensive mice, high salt-induced cardiac hypertrophy and interstitial fibrosis were avoided when AT1 receptors or ACE were pharmacologically inhibited [27]. Our data showing an augmented sensitivity of the cardiovascular system to angiotensin II, as well as the reduced hypotensive effect of the AT1 receptor antagonist losartan, help us to understand the efficacy of AT1 receptor antagonists and ACE inhibitors against the deleterious effects associated with high salt intake, since they reveal that, regardless of the reduced amounts of plasma angiotensin II found in these animals and the maintenance of systemic arterial pressure at normal values, the cardiovascular system remains fully responsive to angiotensin II.

Activation of angiotensin II AT1 receptors was previously associated with superoxide production, a mechanism that may contribute with the development of hypertension [35]. Interestingly, vessels isolated from both Sprague–Dawley rats fed for 3 days with 4% NaCl chow [47] and C57BL/6J mice fed a 7% NaCl chow for 4 weeks [31] displayed reduced nitric oxide production and increased superoxide generation, that was mediated at least in part by uncoupled endothelial nitric oxide synthase [32], and was enhanced in endothelial nitric oxide synthase knockout mice [25]. In our study we found reduced plasma levels of nitric oxide metabolites (nitrite/nitrate, as measured by Griess reaction) in rats fed with 8% NaCl chow (data not show). Although we have not explored the involvement of superoxide and nitric oxide production in our findings, it is reasonable considering that an unbalanced production of these mediators after the continuous high salt intake would reduce the influence of basal and stimulated nitric oxide against vasoconstrictor events, contributing to the increasing peak and duration of the pressor effects of angiotensin II described in this study. Nevertheless, based in the results obtained in both anesthetized and conscious rats, these changes were not enough to induce hypertension in healthy rats even after a period of six weeks under high salt intake, although deleterious changes in vascular function and structure, which may precede the development of hypertensive states, cannot be discarded. The role of AT1 receptors, NADPH-oxidase, superoxide generation and oxidative stress for the vascular function of animals subjected for high salt intake deserves further investigation.

5. Conclusions

In summary, despite the finding that RAAS appears to be less active in healthy rats (neither salt-sensitive nor hypertensive) subjected to high salt intake, as demonstrated by reduced plasma levels of both angiotensin II and aldosterone, our data, obtained in vivo after 6 weeks of high salt ingestion, suggest that the systemic ability of ACE to convert angiotensin I to angiotensin II is maintained or even increased in healthy rats subjected to high amounts of sodium. This is demonstrated by the enhanced pressor effects induced by intravenous administration of angiotensin I, the physiological precursor of angiotensin II, as well as the reduced effects of bradykinin, an endogenous vasodilator able to stimulate nitric oxide production in endothelial cells. This study also shows that the increased ability of the cardiovascular system to convert angiotensin I to angiotensin II is associated with enhanced effects of angiotensin II on arterial pressure. This may explain why rodents usually do not display hypotension, in spite of reduced plasma levels of both angiotensin II and aldosterone. On the other hand, the enhanced sensitivity to angiotensin II that is mediated, at least in part, by increased expression of AT1 receptors and reduced expression of AT2 receptors, may render the entire cardiovascular system susceptible to the deleterious effects mediated by angiotensin II. Importantly, the reduced effects of losartan in rats fed high salt diet indicates that the up-regulation of AT1 receptors in the vascular system contributes to the maintenance of the regulatory effects of endogenous angiotensin II on the arterial pressure of these animals, and may play a role in the vascular dysfunction associated with high salt intake in normotensive animals. If extrapolated to humans, our study emphasizes the potential involvement of the renin–angiotensin–aldosterone system in the risks associated with high salt consumption, even in the absence of systemic hypertension. The systemic, local and cellular consequences involved in this process deserve further investigation.

Abbreviations

RAAS

renin–angiotensin–aldosterone system

ACE

angiotensin-converting enzyme

MAP

mean arterial pressure