The type 2 angiotensin receptor (AT2R) has been suggested to counterbalance the type 1 angiotensin receptor (AT1R) in the central regulation of blood pressure and sympathetic tone. In the present study we investigated the blood pressure responses to stimulation of central AT2Rs by the selective agonist Compound 21 in conscious spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKY rats). We also assessed the impact on noradrenaline [norepinephrine (NE)] plasma levels, autonomic function, spontaneous baroreflex sensitivity, and the possible involvement of the nitric oxide (NO) pathway and the AT1Rs. Chronic intracerebroventricular Compound 21 infusion lowered blood pressure and NE plasma levels in both rat strains. The night-time hypotensive effect was greater in SHRs compared with WKY rats. Compound 21 improved spontaneous baroreflex sensitivity more in SHRs than in WKY rats. These effects were abolished by co-administration of the AT2R antagonist PD123319 or the NO synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Central AT1R blockade did not enhance the hypotensive response to Compound 21. Chronic selective stimulation of central AT2Rs lowers blood pressure through sympathoinhibition, and improves spontaneous baroreflex sensitivity more in SHRs than in WKY rats. These responses appear to require a functioning central NO pathway, but are not modified by central AT1R blockade. Collectively, the data demonstrate specific beneficial effects of stimulation of central AT2Rs in hypertension associated with increased sympathetic tone, and suggest that central AT2Rs may represent a potential new therapeutic target for the treatment of neurogenic hypertension.

CLINICAL PERSPECTIVES

  • Collectively, the data from our study support a role for AT2Rs as an important element in the beneficial arm of the RAS. We provide the first evidence that chronic selective central AT2R stimulation attenuates hypertension and improves autonomic dysfunction and impaired baroreflex sensitivity in SHRs.

  • In contrast to peripheral AT2Rs, for which there is now abun-dant evidence that their stimulation does not result in consistent blood-pressure-lowering effects, the present study suggests that centrally acting AT2R agonists may have significant blood-pressure-lowering effects provided that they can cross the blood–brain barrier.

  • Further research into a better understanding of the location-, age- and disease-dependent roles of the AT2Rs is warranted before AT2R agonists can be brought to the clinic.

INTRODUCTION

Angiotensin II (AngII), the key player in the renin–angiotensin system (RAS), mediates its effects mainly via the type 1 (AT1R) and type 2 (AT2R) angiotensin receptors [1]. AT1Rs are widely distributed throughout the body and mediate the classic cardiovascular effects of AngII, such as vasoconstriction, sodium retention, promotion of inflammatory responses, vascular smooth muscle cell proliferation and cardiac hypertrophy [1]. Current evidence suggests that the AT2R plays a counter-regulatory role opposing the AT1R-mediated actions by promoting vasodilatation, natriuresis, and anti-inflammatory, anti-proliferative and anti-fibrotic responses [2]. Activation of the so-called protective arm of the RAS through stimulation of the AT2Rs has shown therapeutic potential in protecting against myocardial and brain injury [3]. Although AT2R stimulation can cause vasodilatation ex vivo, peripheral AT2R stimulation does not translate into a significant antihypertensive effect in vivo, probably due to the dominating AT1R-mediated vasoconstrictive tone [4].

The key role of the brain's RAS, and in particular the AT1Rs, in the regulation of blood pressure and sympathetic tone is well established [5,6]. It is well known that brain AngII, acting through AT1Rs, increases mean arterial pressure (MAP) and sympathetic nerve activity, but the possible role(s) of the central AT2Rs in cardiovascular regulation remains incompletely understood. Recent evidence suggests that the AT2Rs may also have a role in blood pressure regulation through sympathomodulation [7,8]. Early investigations showed that intracerebroventricular injection of AngII evoked a larger increase in blood pressure in AT2R-knockout mice compared with wild-type mice, linking the central AT2Rs to blood pressure regulation and suggesting a counter-regulatory role for brain AT2Rs [9,10]. In addition, over-expression of AT2Rs in the rostral ventrolateral medulla (RVLM), a primary brainstem nucleus related to the control of sympathetic outflow, reduced blood pressure and urinary noradrenaline [norepinephrine (NE)] excretion in normal Sprague–Dawley rats [11].

The availability of the non-peptide AT2R agonist Compound 21 (C21) [12,13] offers the possibility of selectively and specifically investigating AT2R-mediated effects. C21 was reported to have cardio-, cerebro- and nephro-protective, as well as anti-inflammatory, effects. Its effect on vascular tone is complex and depends on experimental conditions [13]. We are aware of only one previous study, in conscious normotensive Sprague–Dawley rats, that used central administration of C21 to investigate the effect of selective brain AT2R stimulation on blood pressure [14]. Central infusion of C21 in this rat strain decreased blood pressure and night-time urinary NE excretion, suggesting a central inhibitory influence of C21 on sympathetic outflow [14]. In previous studies in our laboratory we were unable to detect direct blood-pressure-lowering effects after an intravenous bolus injection or infusion of different doses of C21, even during AT1R blockade [15], indicating a lack of consistent blood-pressure-lowering effect after peripheral C21 administration. Currently, it is unknown whether central AT2R stimulation decreases blood pressure and sympathetic tone in the hypertensive setting.

In the present study, we first aimed to confirm that in vivo chronic central stimulation of AT2Rs by C21 reduces blood pressure in Wistar Kyoto (WKY) rats, another normotensive rat strain. Our main objective was to investigate the responses evoked by chronic intracerebroventricular infusion of C21 in spontaneously hypertensive rats (SHRs), a model of neurogenic hypertension. We also explored the potential mechanism(s) underlying the impact of C21 on blood pressure by investigating the effects of C21 on autonomic function and spontaneous baroreflex reflex sensitivity (SBRS). As nitric oxide (NO) generated within the central nervous system (CNS) is known to interact with the brain RAS, including the AT2Rs, to modulate the sympathetic nerve activity and blood pressure, we also determined the possible role of the NO pathway in the responses evoked via central AT2R activation by C21 [6,8,1620].

MATERIALS AND METHODS

Animals

Male WKY rats and SHRs (Charles River Laboratories), aged 14 weeks, were housed individually in a temperature- (range 20–26°C) and humidity-controlled (range 30–70%) facility under a 12-h light/12-h dark cycle, maintained on normal rat diet with free access to tap water. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Boston University School of Medicine Institutional Animal Care and Use Committee.

Surgical procedures

A radiotelemetry device (PA-C40, DSI) was implanted using telemetry probe implantation into the abdominal aorta via the left femoral artery under ketamine anaesthesia (intraperitoneal ketamine 30 mg/kg and xylazine 3 mg/kg).

After telemetry implantation and surgical recovery (5–7 days), animals were anaesthetized (intraperitoneal ketamine 30 mg/kg in combination with intraperitoneal xylazine 3 mg/kg) and stereotaxically implanted with a stainless steel cannula into the right lateral cerebral ventricle (Plastics One), which was connected via Silastic tubing to an osmotic mini-pump (Model 2004, Durect Corp.) for intracerebroventricular infusion [21].

Experimental protocols

Responses to intracerebroventricular infusion of the selective AT2R agonist C21 were assessed in WKY rats and SHRs.

After completion of all surgical procedures and surgical recovery (5–7 days), the baseline blood pressure was recorded using telemetry on a scheduled sampling for 10 s at 10-min intervals over a 7-day period, in WKY rats and SHRs. The mean of these values for every 24-h period was calculated. For the day- and night-time measurements, the mean values for the 12-h light cycle and 12-h dark cycle, respectively, were calculated. To compare different treatments, changes in the MAP were calculated as the change in blood pressure compared with day 7 of the baseline period. After 7 consecutive days of baseline measurements in animals receiving an intracerebroventricular saline-vehicle infusion, rats were randomly assigned (n=6–8 per group) into treatment groups. WKY rats were followed up for an additional 7-day treatment period and SHRs for a 14-day treatment period because their blood pressure continued to increase over the whole 14-day period, in contrast to WKY rats. All compounds were dissolved in isotonic saline and infused intracerebroventricularly at a rate of 0.25 μl/h. Comparisons between WKY rats and SHRs were made on the same treatment days. The 500 ng dose of C21 was selected based on a previous study showing a hypotensive response in normotensive Sprague–Dawley rats [14]. In addition, lower doses were also investigated to detect the minimal effective dose of 20 ng of C21. Co-infusion treatments with the AT1R blocker losartan (10 μg/h) and the NO synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (50 μg/h) were given through the same osmotic mini-pump. The protocol is shown in Supplementary Figure S1. Treatment groups and their doses are depicted in Table 1.

Table 1
Overview of the treatment groups in WKY rats and SHRs
WKY ratsSHRs
Saline-vehicle control Saline-vehicle control 
C21 (2 ng/h)  
C21 (10 ng/h)  
C21 (20 ng/h) C21 (20 ng/h) 
C21 (500 ng/h) C21 (500 ng/h) 
C21 (500 ng/h)+PD123319 (500 ng/h) C21 (500 ng/h)+PD123319 (500 ng/h) 
C21 (20 ng/h)+PD123319 (20 ng/h) C21 (20 ng/h)+PD123319 (20 ng/h) 
PD123319 (20 ng/h) PD123319 (20 ng/h) 
C21 (500 ng/h)+losartan (10 μg/h) C21 (500 ng/h)+losartan (10 μg/h) 
C21 (20 ng/h)+losartan (10 μg/h) C21 (20 ng/h)+losartan (10 μg/h) 
C21 (20 ng/h)+L-NAME (50 μg/h) C21 (20 ng/h)+L-NAME (50 μg/h) 
L-NAME (50 μg/h) L-NAME (50 μg/h) 
WKY ratsSHRs
Saline-vehicle control Saline-vehicle control 
C21 (2 ng/h)  
C21 (10 ng/h)  
C21 (20 ng/h) C21 (20 ng/h) 
C21 (500 ng/h) C21 (500 ng/h) 
C21 (500 ng/h)+PD123319 (500 ng/h) C21 (500 ng/h)+PD123319 (500 ng/h) 
C21 (20 ng/h)+PD123319 (20 ng/h) C21 (20 ng/h)+PD123319 (20 ng/h) 
PD123319 (20 ng/h) PD123319 (20 ng/h) 
C21 (500 ng/h)+losartan (10 μg/h) C21 (500 ng/h)+losartan (10 μg/h) 
C21 (20 ng/h)+losartan (10 μg/h) C21 (20 ng/h)+losartan (10 μg/h) 
C21 (20 ng/h)+L-NAME (50 μg/h) C21 (20 ng/h)+L-NAME (50 μg/h) 
L-NAME (50 μg/h) L-NAME (50 μg/h) 

SBRS was calculated using the sequence method [22] during the baseline period on days 2 and 7 for WKY rats and SHRs, and during the intracerebroventricular treatment period on days 9 and 14 for WKY rats, and on days 9, 14 and 21 for SHRs (HemoLab Software version 16.0). A time-dependent analysis was done. Radiotelemetry data were collected, stored and analysed using Dataquest A.R.T. 4.33 software (DSI). For further investigation of possible changes in autonomic nervous system activity evoked by C21 infusion, acute systemic atropine and propranolol challenges were carried out. After a 30-min continuous measurement of baseline heart rate (HR) and MAP via radiotelemetry, an intraperitoneal bolus of atropine (1 mg/kg) or propranolol (2 mg/kg) was administered and peak changes in HR and MAP were recorded. These studies were conducted for WKY rats on day 14 and for SHRs on day 21.

In certain groups, at the end of the protocol (day 15 for WKY rats and day 22 for SHRs) plasma was collected and stored for subsequent measurement of plasma NE concentrations. After conscious decapitation, trunk blood was collected in EDTA tubes and immediately centrifuged for 15 min at 1000 g at 4°C, and the supernatant was stored at −80°C. The harvesting was done in the morning at the same time point for all animals, synchronized with the previous SBRS measurements.

In the saline-vehicle control experiments and the experiments involving losartan infusion, after measurement of baseline water intake for 30 min, animals received an intracerebroventricular injection of AngII (100 ng) and the dipsogenic response was recorded over 30 min [23] to confirm the blockade of the central AT1Rs.

After completing the above-described protocol, rats from the saline-vehicle control group and the C21 (20 ng/h) group were housed in individual metabolic cages for a 24-h period (model 18cv, Fenco) with external food containers and water bottles, for WKY rats on day 14 and SHRs on day 21. Metabolic cages were equipped with a double-fine mesh screen which allowed separation of food and faeces from urine; the urine was collected in vials that contained a layer of mineral oil to prevent urine evaporation. Measurements were made for food and water intake, and urine output over a 24-h period, enabling calculation of sodium and water balance [24].

The plasma NE concentration was determined by ELISA (IBL America). For all groups, the ELISA was performed in duplicate and a mean of the results was taken. The plasma samples were not pooled from animals, each sample being from an individual rat. Urinary sodium content was determined by flame photometry (model 943, Instrumentation Laboratories) and urinary osmolality was measured by a vapour pressure osmometer (Vapro 5500, Wescor Inc.). Free water clearance and 24-h sodium excretion were calculated. Urinary sodium excretion (mmol/24 h) equals 24-h urine output (ml) times urinary sodium concentration (mmol/l). Free water clearance (CH2O) was calculated as the difference between the rate of urine volume (ml) per 24 h and the osmolar clearance [24].

Drugs

Losartan, PD123319 and AngII were purchased from Sigma-Aldrich Co. and L-NAME was from Santa Cruz Biotechnology. C21 was provided by Vicore Pharma AB. Doses were selected based on previous studies [14,25,26].

Statistical analysis

Data are expressed as means±S.E.M. Data were analysed using Student's t tests, ANOVA and appropriate post-hoc analyses. Differences occurring between treatment groups (e.g. C21 vs control) were assessed using a two-way repeated-measure ANOVA, followed by Bonferroni's post-hoc test, to compare variations among the groups. P was set at 0.05. All calculations and graphs were obtained by using GraphPad Prism 4.03.

RESULTS

AT2R-mediated blood pressure and heart rate responses to intracerebroventricular C21

WKY rats

Mean blood pressure and heart rate values (average over 24 h) in WKY rats during the 7-day period of baseline measurements were 110±2 mmHg and 360±4 beats/min; these values remained constant. In the control group, during saline-vehicle infusion over the 14-day study period, these values also remained constant (Figure 1A and see Supplementary Figure S2A).

Change in the MAP in WKY rats
Figure 1
Change in the MAP in WKY rats

Results are shown as means±S.E.M. (n=6–8 per group). §P<0.05, §§P<0.01, §§§P<0.001 compared with C21 at 20 ng; *P<0.05, **P<0.01, ***P<0.001 compared with saline-vehicle control (multiple comparisons, two-way ANOVA). #P<0.05, ##P<0.01, ###P<0.001 compared with baseline values on day 7 (ANOVA for repeated measures).

Figure 1
Change in the MAP in WKY rats

Results are shown as means±S.E.M. (n=6–8 per group). §P<0.05, §§P<0.01, §§§P<0.001 compared with C21 at 20 ng; *P<0.05, **P<0.01, ***P<0.001 compared with saline-vehicle control (multiple comparisons, two-way ANOVA). #P<0.05, ##P<0.01, ###P<0.001 compared with baseline values on day 7 (ANOVA for repeated measures).

Intracerebroventricular infusion of C21 during the 7-day treatment period at doses of 20 ng/h and 500 ng/h significantly lowered MAP by −6.1±0.6 and −5.6 ±0.9 mmHg, respectively, compared with pre-treatment on day 7, and by −6.8±0.7 and −6.3±0.5 mmHg, respectively, compared with saline-vehicle control group values on day 14 (P<0.05) (Figure 1A and see Supplementary Figure S3A). Lower doses of C21 (2 ng/h, 10 ng/h) did not alter MAP (see Supplementary Figure S3B).

Co-infusion of the AT2R antagonist PD123319 with C21 (20 ng/h and 500 ng/h) abolished the C21-evoked decrease in MAP (Figure 1A, and see Supplementary Figures S2A and S3A). PD123319 alone did not significantly change MAP (see Supplementary Figure S3C). The HR did not change in any of these experimental groups (see Supplementary Figure S4A).

SHRs

MAP increased progressively in SHRs over the 7-day baseline period from day 0 onwards, with, however, some variation in the magnitude of the blood pressure increase between individual animals. We therefore considered, in each group, the average MAP measured at day 7, i.e. immediately before infusion of the test compounds versus the saline vehicle, as a baseline. These baseline values for MAP and HR were 153±5 mmHg and 355±4 beats/min. The MAP increased progressively from baseline during the 14-day period in the saline-vehicle control SHRs by +11.7±2.9 mmHg to a value of 164±5 mmHg (P<0.01) (Figure 2A and see Supplementary Figure S5A).

Change in the MAP in SHRs
Figure 2
Change in the MAP in SHRs

Results are shown as means±S.E.M. (n=6–8 per group). §P<0.05, §§§P<0.001 compared with C21 at 20 ng; *P<0.05, **P<0.01, ***P<0.001 compared with saline-vehicle control; £P<0.05, ££P<0.01, £££P<0.001 compared with C21 at 20 ng+L-NAME (multiple comparisons, two-way ANOVA). ##P<0.01, ###P<0.001 compared with baseline values on day 7 (ANOVA for repeated measures).

Figure 2
Change in the MAP in SHRs

Results are shown as means±S.E.M. (n=6–8 per group). §P<0.05, §§§P<0.001 compared with C21 at 20 ng; *P<0.05, **P<0.01, ***P<0.001 compared with saline-vehicle control; £P<0.05, ££P<0.01, £££P<0.001 compared with C21 at 20 ng+L-NAME (multiple comparisons, two-way ANOVA). ##P<0.01, ###P<0.001 compared with baseline values on day 7 (ANOVA for repeated measures).

Intracerebroventricular infusion of C21 for a 14-day period at doses of 20 ng/h and 500 ng/h prevented this spontaneous blood pressure increase and reduced MAP from baseline values recorded on day 7 of saline infusion by −6.1±1.6 and −3.8 ±1.5 mmHg, respectively. After 7 days of C21 infusion, at day 14 of the protocol, the difference in MAP compared with the saline-vehicle control group was −9.0±1.7 and −5.3±1.4 mmHg, respectively, and the decrease in MAP was highly significant after 14 days of C21 infusion, at day 21 of the protocol, −18.0±2.0 and −15.5±1.6 mmHg, respectively (P<0.001) (Figure 2A and see Supplementary Figure S6A). The hypotensive effect of intracerebroventricular C21 infusion was abolished by PD123319 co-infusion (P<0.001 vs C21 alone) (Figure 2A, and see Supplementary Figures S5A and S6A). PD123319 alone did not significantly change MAP compared with the saline control group (see Supplementary Figure S6C). No significant HR changes were observed in these SHR experiments (see Supplementary Figure S7A). The magnitude of the hypotensive response (24 h average) after the 7-day infusion of C21 (20 ng/h) (compared with saline for the same period) tended to be slightly greater in SHRs than in WKY rats, but the difference did not reach statistical significance. However, after 14 days of infusion in SHRs, the blood pressure-lowering effect was more pronounced and significantly greater than observed after 7 days, in both WKY rats and SHRs (P<0.001).

Night-time versus daytime blood pressure

We performed an additional analysis of the day- versus night-time blood pressures. In both strains, night-time blood pressure was significantly higher than daytime blood pressure, both under saline infusion [night vs day MAP (mmHg): WKY rats +5.0±0.3; SHRs +5.8±0.5; P<0.001] and during C21 infusion (20 ng/h) [night vs day MAP (mmHg): WKY rats +4.9±0.3; SHRs +5.4±0.5; P<0.001]. The magnitude of the blood pressure reduction induced by a 7-day C21 infusion was similar in the two strains during the daytime [C21 evoked a peak change in MAP (mmHg): WKY rats −5.5±0.6, SHRs −5.4±1.5]. However, during the night-time, intracerebroventricular C21 for 7 days reduced blood pressure significantly more in SHR than in WKY rats from the baseline values [C21 evoked peak change in night-time MAP (mmHg): WKY rats −8.2±0.8 vs SHRs −12.6±1.9; P<0.01]. In SHRs, the magnitude of the night-time hypotensive responses to intracerebroventricular C21 for 7 or 14 days was significantly greater than during daytime, after both 7 (P<0.05) and 14 (P<0.001) days; a similar but statistically insignificant trend was seen in WKY rats (Table 2).

Table 2
Change in mean arterial blood pressure (MAP; mmHg) in Wistar Kyoto rats (WKY) (above) and Spontaneously Hypertensive rats (SHRs) (below).

Data are shown as mean±SEM (n=6–8 per group).

WKY
D14C21 icvSaline icv
24 h −6.8±0.7* +0.7±0.1    
Night −8.2±0.8** +0.8±0.1    
Day −5.5±0.6* +1.1±0.2    
SHR     
D14C21 icvSaline icvD21C21 icvSaline icv
24 h −9.0±1.7# +3.9±1.1 24 h −18.0±2.0### +11.6±2.1 
Night −12.6±1.9# $$ +5.2±0.4 Night −24.8±2.2### +14.6±1.5 
Day −5.4±1.5# +3.8±0.3 Day −11.3±1.8###  
WKY
D14C21 icvSaline icv
24 h −6.8±0.7* +0.7±0.1    
Night −8.2±0.8** +0.8±0.1    
Day −5.5±0.6* +1.1±0.2    
SHR     
D14C21 icvSaline icvD21C21 icvSaline icv
24 h −9.0±1.7# +3.9±1.1 24 h −18.0±2.0### +11.6±2.1 
Night −12.6±1.9# $$ +5.2±0.4 Night −24.8±2.2### +14.6±1.5 
Day −5.4±1.5# +3.8±0.3 Day −11.3±1.8###  

D: day. *P<0.05, **P<0.01 vs WKY rat saline control, #P<0.05, ###P<0.001 vs SHR saline control, $$P<0.01 vs WKY rat C21.

Effect of intracerebroventricular C21 infusion on AT2R-mediated changes in autonomic and renal function

NE plasma levels

In control intracerebroventricular vehicle-saline-infused rats, plasma NE levels were significantly lower in WKY rats (217.0±19.4 pg/ml) than in SHRs (317.2±14.7 pg/ml; P<0.05). C21 infusion (20 ng/h) significantly decreased NE plasma concentration compared with saline infusion in WKY rats to 180.1±13.6 pg/ml (P<0.05) and in SHRs to 262.9 ±19.8 pg/ml (P<0.05). In both strains, PD123319 alone had no effect on NE (n=3, results not shown) but abolished the decreases in NE plasma concentration evoked by intracerebroventricular C21 infusion (Figure 3).

NE plasma levels in WKY rats and SHRs
Figure 3
NE plasma levels in WKY rats and SHRs

Results are shown as means±S.E.M. (n=6–8 per group). *P<0.05 compared with corresponding saline control. #P<0.05 comparing saline control in SHRs with corresponding saline control in WKY rats.

Figure 3
NE plasma levels in WKY rats and SHRs

Results are shown as means±S.E.M. (n=6–8 per group). *P<0.05 compared with corresponding saline control. #P<0.05 comparing saline control in SHRs with corresponding saline control in WKY rats.

Autonomic function

C21 infusion had no effect on the increase in HR evoked by intraperitoneal bolus atropine in either WKY rats (vehicle +119.7±11.0 vs C21 +114.0±12.5 beats/min, not significant (NS)) or in SHRs (vehicle +102.2±11.3 vs C21 +107.1±5.4 beats/min, NS). The bradycardic response to an intraperitoneal bolus of propranolol was significantly attenuated in the C21-treated animals (WKY rats: vehicle −49.7±3.0 vs C21 −25.0±3.2 beats/min, P<0.05; SHRs: vehicle −68.2±2.4 vs C21 −36.3±4.1 beats/min, P<0.05). PD123319 co-infusion abolished this effect of C21 (Figure 4).

Peak change in HR in WKY rats (upper panels) and SHRs (lower panels) after intraperitoneal injection of atropine and propranolol at the end of C21 infusion
Figure 4
Peak change in HR in WKY rats (upper panels) and SHRs (lower panels) after intraperitoneal injection of atropine and propranolol at the end of C21 infusion

Results are shown as means±S.E.M. (n=6–8 per group). ***P<0.001 compared with corresponding saline control. ###P<0.001 comparing saline control in SHRs with corresponding saline control in WKY rats on day 7.

Figure 4
Peak change in HR in WKY rats (upper panels) and SHRs (lower panels) after intraperitoneal injection of atropine and propranolol at the end of C21 infusion

Results are shown as means±S.E.M. (n=6–8 per group). ***P<0.001 compared with corresponding saline control. ###P<0.001 comparing saline control in SHRs with corresponding saline control in WKY rats on day 7.

SBRS

SBRS, as measured on days 2 and 7 of the baseline period under saline-vehicle infusion, was significantly impaired in SHRs compared with WKY rats [SBRS (ms/mmHg) saline infusion: WKY rats, day 2, 2.6±0.3, day 7, 2.5±0.4 vs SHRs, day 2, 2.0±0.1, day 7, 1.8±0.2; both P<0.05) (Figures 5A and 5E).

SBRS in WKY rats (A–D) and SHRs (E–H)
Figure 5
SBRS in WKY rats (A–D) and SHRs (E–H)

Results are shown as means±S.E.M. (n=6–8 per group). **P<0.01 compared with corresponding saline control, day 2 vs day 9, day 7 vs day 14, day 7 vs day 21. #P<0.05 comparing saline control in SHRs with corresponding saline control in WKY rats. $$$P<0.001 comparing change in SBRS in SHRs with corresponding change in WKY rats.

Figure 5
SBRS in WKY rats (A–D) and SHRs (E–H)

Results are shown as means±S.E.M. (n=6–8 per group). **P<0.01 compared with corresponding saline control, day 2 vs day 9, day 7 vs day 14, day 7 vs day 21. #P<0.05 comparing saline control in SHRs with corresponding saline control in WKY rats. $$$P<0.001 comparing change in SBRS in SHRs with corresponding change in WKY rats.

C21 infusion (20 ng/h) immediately and significantly increased SBRS in both strains; this effect was maintained throughout the infusion period [SBRS (ms/mmHg) C21 at 20 ng/h infusion; WKY rats day 9, 3.6±0.3, day 14, 3.7±0.4, both P<0.01 vs baseline; SHRs day 9, 3.2±0.2, day 14, 3.4±0.2, day 21, 3.7±0.1, all P<0.01 vs baseline) (Figures 5A and 5E). The improvement in SBRS on day 14 was more pronounced in SHRs than in WKY rats (84% vs 46%, P<0.001). A higher-concentration C21 infusion (500 ng/h) also significantly increased SBRS [SBRS (ms/mmHg) C21 500 ng/h infusion: WKY rats day 9, 3.9±0.4, day 14, 3.8±0.5, both P<0.01; SHRs day 9, 3.0±0.6, day 14, 3.0±0.5, day 21, 3.2±0.5, all P<0.01) (see Supplementary Figure S8). The C21-induced increase in SBRS was abolished by co-infusion of PD123319 in both strains (Figures 5B and 5F), whereas infusion of PD123319 alone had no significant effect on SBRS.

Fluid and electrolyte homoeostasis

C21 infusion did not alter the sodium balance or free water clearance in WKY rats or SHRs. We observed no difference in sodium excretion (WKY rats: vehicle −0.67±0.2 vs C21 −0.77±0.2 mmol/day, NS; SHRs: vehicle −0.54±0.1 vs C21 −0.47±0.1, NS) or free water clearance (WKY rats: vehicle −24.3±4.5 vs C21 −25.3±4.2 ml/day, NS; SHRs: vehicle −26.5±4.2 vs C21 −28.1±4.2, NS) after C21 infusion (see Supplementary Figure S9).

Impact of intracerebroventricular AT1Rs and NO on AT2R-mediated physiological effects

WKY rats

Co-infusion of the AT1R antagonist losartan, in a dose (10 μg/h) that abolished the dipsogenic response to intracerebroventricular injection of AngII seen in control WKY rats and SHRs (see Supplementary Figure S10), with C21 (20 and 500 ng/h) did not further enhance the MAP-lowering effect seen with C21 alone (20 and 500 ng/h) (see Figure 1B, and Supplementary Figures S2B and S3D).

L-NAME infusion alone significantly increased the MAP from baseline values by +11.8±1.7 mmHg (P<0.001) (see Figure 1C and Supplementary Figure S2C) and decreased the HR from baseline by −24.4±4.7 beats/min (P<0.05) (see Supplementary Figure S4B) without altering the SBRS (see Figure 5D). Co-infusion of L-NAME and C21 abolished the hypotensive effect of C21, evoking an increase in the MAP from baseline values by +8.4±0.9 mmHg (P<0.01 vs C21 alone) (see Figure 1C and Supplementary Figure S2C) and also abolished C21-induced increases in SBRS (see Figure 5C).

SHRs

As in the WKY rats co-infusion of the AT1R antagonist losartan (10 μg/h) with C21 (20 and 500 ng/h) did not further enhance the MAP-lowering effect seen with C21 alone (20 and 500 ng/h) in SHRs (see Figure 2B and Supplementary Figures S5B and S6B).

L-NAME infusion alone significantly increased the MAP from baseline by +38.7±2.5 mmHg (P<0.05) (see Figure 2C and Supplementary Figure S5C) and tended to reduce the HR. L-NAME (50 μg/h) co-infusion blocked the effect of C21 (20 ng/h) and increased the MAP from baseline by +37.1±3.2 mmHg (P<0.05 vs C21 alone) (see Figure 2C and Supplementary Figure S5C) and significantly reduced the HR from baseline by −16.0±2.8 beats/min (P<0.05) (see Supplementary Figure S7B). Although MAP was still significantly different (P<0.05) from day 10 until day 13 between the groups C21 at 20 ng+L-NAME and L-NAME alone, MAP and HR with co-infusion of C21+L-NAME were no different from the corresponding values with L-NAME infusion alone at day 21. C21-induced increases in SBRS were again abolished by co-infusion of L-NAME (see Figure 5G); intracerebroventricular infusion of L-NAME alone did not alter the SBRS (see Figure 5H).

DISCUSSION

The major novel finding of the present study is that central chronic stimulation of the AT2R by the selective non-peptide AT2R agonist C21 evoked a sustained decrease in blood pressure not only in normotensive but also in spontaneously hypertensive rats (SHRs) in vivo, and that this hypotensive response is associated with sympathoinhibition and increased SBRS. These data further demonstrate that there is a differential response to C21 between SHRs and WKY rats in many parameters.

It is well established that brain AngII induces tonic sympatho-excitatory effects, resulting in blood pressure increases through stimulation of central AT1Rs. However, the possible role of brain AT2Rs in blood pressure control is less well understood, although current evidence suggests that in the RVLM they may mediate sympathoinhibitory effects [6,11]. In the present study, we observed that chronic intracerebroventricular infusion of C21, at doses of 20 or 500 ng/h, consistently lowered blood pressure and plasma NE concentrations in normotensive WKY rats. Recent in vitro experiments suggested that C21 may, similar to most other drugs, induce non-specific effects in concentrations >1 μM [27]. However, the central blood-pressure-lowering and sympatholytic effects observed in the present study were observed at much lower concentrations of C21 (0.04 μmol/l). Moreover, these effects were both abolished by concomitant infusion of PD123319, administered in a dose known to selectively block the AT2Rs [28], and not reaching the high concentration at which it would be expected also to block AT1Rs, confirming that these responses were probably AT2R-mediated.

The results obtained in WKY rats validate and extend earlier findings conducted in male Sprague–Dawley rats [14] that AT2R activation lowers blood pressure in normotensive rat phenotypes. These authors also reported a reduction in night-time urinary NE excretion, supporting our finding of a sympathoinhibitory response to chronic central AT2R stimulation. It is of interest to note that, whereas the effects of C21 were abolished by PD123319, indicating that exogenous stimulation of brain AT2Rs in normotensive rats results in sympathoinhibition, chronic infusion of PD123319 alone had no effect on the MAP or NE levels, suggesting that endogenous activation of brain AT2Rs does not appear to contribute significantly to the control of blood pressure and sympathetic tone under basal conditions.

The most important novel observations of the present study relate to the experiments conducted in SHRs. To our knowledge this is the first study investigating responses to chronic central AT2R stimulation through intracerebroventricular infusion of C21 in an in vivo conscious animal model of hypertension. As expected, and in line with the available literature [29], blood pressure progressively increased by almost 12 mmHg in the control SHRs followed for a 21-day period of saline-vehicle infusion. Intracerebroventricular infusion of C21 from day 8 to day 21 completely prevented this progressive blood pressure increase and further lowered MAP to below the baseline levels on day 7. The magnitude of this response was significantly greater than the response seen in either SHRs or WKY rats after 7 days of infusion. As expected, night-time blood pressure was significantly higher than daytime blood pressure in both strains. It is of interest that, whereas the magnitude of the blood pressure reduction induced by C21 was similar in the two strains during the daytime, in SHRs the night-time hypotensive response to C21 was significantly greater than during the daytime. Moreover, at night-time, intracerebroventricular C21 for 7 days reduced blood pressure significantly more in SHRs than in WKY rats, suggesting that brain AT2Rs may be involved in blood pressure control in particular during the night-time in SHRs.

The marked hypotensive response in a model of hypertension induced by specific and selective stimulation of brain AT2Rs observed in the present study is in sharp contrast to the lack of effect on blood pressure by stimulation of peripheral AT2Rs [4]. Indeed, although evidence of AT2R-mediated vasodilatation is available ex vivo, in vivo studies on the possible blood-pressure-lowering effect of peripheral AT2R stimulation in hypertensive animal models have yielded conflicting results, and hypotensive responses were either not detectable or only during co-administration of an AT1 antagonist at a low dose [30,31]. This lack of significant antihypertensive effect has resulted in the conclusion that non-peptide AT2R agonists would not become a new class of antihypertensive drugs. The present study, however, suggests that centrally acting AT2R agonists may have significant blood-pressure-lowering effects provided that they can cross the blood–brain barrier.

Our observation that C21-mediated stimulation of central AT2Rs prevented the spontaneous blood pressure increase in SHRs is in line with the recent demonstration by Blanch et al. [32] that increased expression of AT2Rs in the solitary-vagal complex, a brainstem region important in the control of blood pressure, attenuates the increase in arterial pressure observed in a rat model with two-kidney one-clip renovascular hypertension.

In line with previous evidence indicating that SHRs have a higher sympathetic tone compared with normotensive control rats [33], baseline plasma NE concentrations were higher in SHRs compared with WKY rats. As observed in the WKY rats, the hypotensive response to infusion of C21 in SHRs was also associated with a significant reduction in the plasma concentrations of NE, measured as a surrogate marker of sympathetic tone. Again, both responses were abolished by concomitant infusion of PD123319, confirming the direct involvement of the AT2Rs in these responses. As in the WKY rat experiments, infusion of PD123319 alone had no significant effect on the MAP or plasma NE concentrations. These results suggest that exogenous stimulation of brain AT2Rs prevents the progressive increase in blood pressure in SHRs through a sympathoinhibitory action.

To investigate possible changes in autonomic nervous system activity evoked by C21 infusion further, acute systemic atropine and propranolol challenges were performed in both SHRs and WKY rats. In both strains, the peak tachycardic responses to an intraperitoneal bolus injection of atropine were unaltered by intracerebroventricular C21 infusion, suggesting that exogenous AT2R stimulation did not alter parasympathetic tone. However, the peak bradycardic responses to propranolol were significantly reduced by C21 infusion, in SHRs as well as in WKY rats, and PD123319 abolished this effect. This is in line with the hypothesis of a C21-induced AT2R-mediated decrease in central sympathetic outflow, without a relevant effect on parasympathetic activity.

Another important finding of our study is that exogenous brain AT2R stimulation improved SBRS in both WKY rats and SHRs. Baroreflex dysfunction is an important hallmark of hypertension [34] closely related to sympathetic hyperactivity and activation of the circulating and local RAS [35]. Reduction in baroreflex sensitivity is considered an independent marker of the risk of mortality and major adverse cardiovascular events in hypertensive patients [36]. SHRs are known to exhibit impaired baroreceptor reflex function [37]. Accordingly, in the present study, SBRS measurements were impaired at baseline and in control experiments with saline-vehicle infusion in SHRs compared with WKY rats. Intracerebroventricular infusion of C21 improved the SBRS in both strains rapidly after the start of the infusion, and this was maintained throughout the whole experiment, but the effect in SHRs was significantly greater than in WKY rats. This C21-induced improvement in SBRS was abolished by a concomitant infusion of the selective AT2R antagonist PD123319, confirming that this effect is mediated through this receptor. It is tempting to speculate that the prevention of the progression of hypertension in SHRs induced by central AT2R stimulation, observed in the present study, is, in part, related to this restoration of baroreceptor function. These results also confirm the recent observation that increased expression of AT2Rs in the solitary-vagal complex restores baroreflex function and sympathetic modulation of arterial pressure to normal values in rats with two-kidney one-clip renovascular hypertension, another rat model characterized by baroreceptor impairment and increased sympathetic tone [32].

The possible in vivo role of peripheral AT2R stimulation in renal sodium handling has been extensively studied [2]. AT2R stimulation by intravenous C21 infusion has been reported to promote natriuresis by a direct effect on tubular function [3840]. On the other hand, in AT2-null mice, no changes in natriu-resis were seen, except under additional AngII infusion, where an anti-natriuretic hypersensitivity was observed [9]. With regard to a possible role for central AT2Rs, a transient increase in urine excretion was reported in normotensive rats with over-expression of AT2Rs in the RVLM [11]. We therefore also investigated whether chronic intracerebroventricular infusion would have an effect on sodium balance and free water clearance, but we could not detect any effect of C21 on fluid and electrolyte handling in WKY rats or SHRs. This negative result does not necessarily contradict the observation of Gao et al. [14], because the increase in urine excretion that they observed was transient and short lasting compared with the prolonged over-expression of the RVLM AT2Rs that they generated, and the measurements of sodium balance and free water clearance obtained in the present study were made at the end of the C21 treatment periods (after 7 days for WKY rats and 14 days for SHRs). Furthermore, the same group did not report the effect of C21 infusion on renal parameters in their subsequent study in Sprague–Dawley rats [14].

The interplay between NO and different components of the RAS, including the AT2Rs, has been previously reported [16,41,42]. We therefore also evaluated whether the responses to intracerebroventricular C21 can be affected by central inhi-bition of NO synthase. In line with the previous observation in normotensive Sprague–Dawley rats [14], we demonstrated that in both WKY rats and SHRs the depressor responses to central AT2R stimulation are blocked by intracerebroventricular infusion of the NO synthase inhibitor L-NAME, although we cannot completely exclude the fact that the lack of detectable hypotensive response to C21 under L-NAME infusion might at least in part be related to the very pronounced blood pressure increase induced by L-NAME itself.

In addition, although the hypotensive response to C21 was abolished after 14 days of L-NAME infusion, it was only partly reversed by L-NAME during the first days of co-infusion. Therefore, other mechanisms might also be involved such as direct cross-talk or down-regulation of AT1Rs. AT2Rs have been shown on activation to heterodimerize with AT1Rs, reducing their cell-surface expression [43]. AT2R/AT1R heterodimerization may also affect intracellular AT1R signalling [44,45], resulting in a functional inhibition of the latter and adding to the complexity of AT2R physiopathology. Nevertheless, as AT2R antagonists such as PD123319 have also been reported to antagonize Mas receptor-mediated effects [46], other effects not directly linked to AT2R stimulation, or an interaction with the Mas-related G-protein-coupled receptor, cannot be entirely excluded. However, we also showed that co-infusion of L-NAME abolished the improvement in baroreceptor sensitivity induced by central AT2R stimulation by C21, indicating that these effects of AT2R stimulation require a functional central NO pathway. These results are in line with a recent study in anaesthetized Wistar rats which suggests a facilitatory role for AT2Rs in high-pressure baroreflex regulation that is NO-dependent [16]. Intracerebroventricular infusion of L-NAME alone significantly increased the MAP and reduced the HR (the latter probably through activation of the high pressure baroreflex), in both WKY rats and SHRs, supporting current evidence of the important role for the brain's endogenous NO in the central control of blood pressure [42]. However, no differences were detected when L-NAME infused alone was compared with the combined intracerebroventricular infusion of L-NAME with C21.

We also addressed the possibility of a contribution of the AT1Rs to the central AT2R-mediated responses in the present study, based on the hypothesis that stimulation of both receptors could result in opposing responses. Peripheral AT1R blockade, to functionally antagonize the AT1Rs, has been required to unmask AT2R-mediated vasodilatation in SHRs [30,31]. Therefore, we also investigated the centrally mediated C21 responses when the counterbalancing actions of AT1Rs were blocked with losartan given at a dose known to have no hypotensive effect by itself after central administration [47], but sufficient to block the dipsogenic effect of exogenous AngII. In contrast with the findings reported for the peripheral vasculature, the hypotensive response to chronic intracerebroventricular C21 infusion is not enhanced by additional central AT1R blockade, in either WKY rats or SHRs. In our studies the response to C21 co-infusion at two different doses (20 ng/h and 500 ng/h) with or without losartan did not differ.

In conclusion, the results of the present study provide the first evidence that chronic selective stimulation of the central AT2Rs by the selective non-peptide AT2R agonist C21 induces a vasodepressor response in hypertensive rats, preventing the progressive blood pressure increase normally observed in this animal model. This hypotensive response is associated with lowered NE plasma levels, suggesting a decrease in sympathetic tone, and improvement of the SBRS. The improvement in baroreceptor reflex sensitivity and the hypotensive effect during the night is more pronounced in SHRs than in WKY rats. In addition this study shows that these brain AT2R-mediated responses require a functioning central NO pathway, but are independent of the presence of functioning central AT1Rs. The modulation of blood pressure regulation and the correction of impaired sympathoregulation potentially achievable by balancing the actions of central inhibitory AT2R versus excitatory AT1R effects, through central AT2R stimulation, could open new therapeutic opportunities for diseases characterized by sympathoexcitation. However, it should be stressed that, although many other studies also reported beneficial responses to AT2R stimulation, the opposite has also been reported in pathological conditions or aging animals, depending on the location of the receptor (endothelium or smooth muscle cell), as well as its capacity to heterodimerize with AT1Rs [48].

AUTHOR CONTRIBUTION

Sofie Brouwers co-designed the study protocols, performed experiments, analysed data, wrote the manuscript and obtained funding. Ilse Smolders, Richard D. Wainford and Alain G. Dupont initiated the study project, obtained the necessary funding, co-designed the study protocols, supervised the analysis, and co-authored and edited the manuscript.

S.B.'s and R.D.W.'s collaboration has been supported by the International Society Hypertension New Investigator Committee. S.B. is a member of the International Society of Hypertension Corporate Liaison Committee and the Networking and Mentorship Working Group of the International Society of Hypertension New Investigator Committee. R.D.W. is a member of the International Society of Hypertension Scientific Council and the Recruitment Working Group of the International Society of Hypertension New Investigator Committee.

FUNDING

This research was supported by the Research Council of the Vrije Universiteit Brussel and the National Institutes of Health [grant number R01HL107330, K02HL112718 (to R.D.W.)] S.B. is holder of research fellowship and travel grant of the Flemish Research Fund and a grant from the Horlait-Dapsens Foundation.

Abbreviations

     
  • AngII

    angiotensin II

  •  
  • AT1R

    type 1 angiotensin receptor

  •  
  • AT2R

    type 2 angiotensin receptor

  •  
  • C21

    Compound 21

  •  
  • CNS

    central nervous system

  •  
  • HR

    heart rate

  •  
  • L-NAME

    Nω-nitro-L-arginine methyl ester hydrochloride

  •  
  • MAP

    mean arterial pressure

  •  
  • NE

    noradrenaline (norepinephrine)

  •  
  • NS

    not significant

  •  
  • RAS

    renin–angiotensin system

  •  
  • RVLM

    rostral ventrolateral medulla

  •  
  • SBRS

    spontaneous baroreceptor reflex sensitivity

  •  
  • SHR

    spontaneously hypertensive rat

  •  
  • WKY

    Wistar Kyoto

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Author notes

1

These authors contributed equally to this work.

Supplementary data