Abstract
Vasoconstrictive, proliferative and oxidative effects of angiotensin II (Ang II) are mediated by Ang II type 1 (AT1) receptors. The effects of Ang II via the Ang II type 2 (AT2) receptor subtype (AT2R) are less well defined. Growing evidence shows the existence of cross-talk between the Ang II receptor subtypes, which is revealed by AT1R blockade. Hence, under certain conditions, AT2R may act as an antagonistic system with respect to the AT1R.
The present study was designed to investigate the effects of long-term treatment with the AT1R antagonist losartan on the AT2R-mediated response to Ang II in thoracic aortas isolated from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Untreated animals from both groups were used as controls. The mRNA expression of AT1R and AT2R was measured by reverse transcription-polymerase chain reaction.
During contraction in response to norepinephrine, Ang II induced concentration-dependent relaxation only in aortas isolated from SHR chronically treated with losartan (8 weeks; 30 mg/kg/day in drinking water). These relaxations were inhibited by the selective AT2R blocker PD123319, NG-nitro-L-arginine methyl ester (L-NAME), and B2receptor antagonist HOE-140. Accordingly, nitric oxide (NO) production was increased by Ang II only in the aortas of treated SHR. After AT1R blockade, AT2R mRNA was significantly increased. These findings demonstrate that, in hypertensive rats, chronic AT1R blockade is associated with an inverted vasomotor response to Ang II via AT2R-mediated NO production.
The losartan-unmasked AT2R-vasorelaxation could significantly contribute to the beneficial hemodynamic effects of AT1R blockade. In view of this, our study highlights the importance of the integrated Ang II receptor network, which may help to define further the mechanisms of the well-established vascular protective effects of AT1R blockers. Am J Hypertens 2005;18:493–499 © 2005 American Journal of Hypertension, Ltd.
The roles of two major subtypes of angiotensin (Ang) II receptors, the Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors, have been investigated both in vivo and in vitro.1,2 The AT1 receptor (AT1R) subtype is expressed ubiquitously and is involved in all of the well-known biological functions of Ang II including vasoconstriction, sodium retention, aldosterone secretion, and trophic effects.3 By contrast, the physiologic role of the AT2receptor (AT2R) remains controversial. Experimental evidence suggests that this receptor acts as an antagonistic system with respect to the AT1R, promoting vasorelaxation and growth inhibition.4 The potential vasodilator effect related to AT2R has been repeatedly described.5–9 Previous studies have also suggested that the vasorelaxing effect of AT2R stimulation is mediated through the bradykinin/nitric oxide/cGMP cascade.5–9 Because nitric oxide (NO) is a principal factor involved in the antiatherosclerotic properties of the vessel wall, the stimulation of AT2R may play an important role in both physiologic and pathologic conditions. Long-term administration of AT1R blockers results in a several-fold increase in plasma concentrations of Ang II due to inhibition of the AT1R-mediated negative feedback on renin release and hence a possible overstimulation of AT2R. Accordingly, in vivo, stimulation of AT2R lowers blood pressure (BP) in SHR and in other models.10–13
Interestingly, cross-talk between the two Ang receptor subtypes has been described.14,15 We have recently shown that Ang II downregulates AT2R mRNA expression via the AT1R binding in rat aortic endothelial cells, whereas an increase in the expression of AT2R occurs during AT1R blockade.14 Thus, AT1R blockade may unmask the potential vasorelaxing properties of AT2R.
Although previous studies5–9 indicate that the AT2R may act as a vasorelaxing pathway counter-regulatory to the constrictor action of Ang II via AT1R, direct assessment of the AT2R-mediated vasomotor responses during chronic AT1R blockade is not available. The present study was designed to investigate the AT2R-mediated vasomotor responses to Ang II and its underlying mechanisms in aortas isolated from SHR and normotensive WKY rats during chronic AT1R blockade.
Methods
Animals and experimental protocols
Eighteen male SHR and 18 WKY rats, 12 to 14 weeks of age, were included in the study. Animals were obtained from Charles River Laboratories (Calco, Italy). All procedures were conducted in accordance with the guidelines for the experimental use of animals of the University of Rome “La Sapienza,” where the studies were performed. Rats were housed two per cage in a room in which temperature, humidity, and light were controlled. Both SHR and WKY rats were treated with losartan (30 mg/kg/day in drinking water) for 8 weeks. Untreated animals of both strains were used as controls. Systolic BP values and heart rate were measured noninvasively using the tail-cuff method in control and treated animals before treatment and after 4 and 8 weeks of treatment. At the end of the 8-week period, both control and treated rats were anesthetized with thiopental, 50 mg/kg intraperitoneally (Abbott Laboratories Diagnostic Division, Chicago, IL). After immediate removal, the thoracic aorta was placed in cold (4°C) modified Krebs-Ringer bicarbonate solution (control solution, mmol/L: NaCl 118.6, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.1, calcium EDTA 0.026, glucose 10.1) and cleaned of adhering connective tissue. The isolated aortas were either cut into rings for vascular reactivity study and for determination of NO production or were frozen in liquid nitrogen and kept at −80°C until isolation of total RNA and reverse transcription-polymerase chain reaction (RT-PCR) were performed.
Organ chamber experiments
Aortic rings (3 to 4 mm in length) were connected to isometric force transducers (Grass, AstroMed, West Warwick, RI), suspended in an organ chamber filled with 25 mL of control solution (37°C; pH 7.4), and bubbled with 95% O2/5% CO2. Isometric tension was recorded continuously. After a 30-min equilibration period, rings were gradually stretched to the optimal point of their length-tension curve (2.5 ± 0.2 g) as determined by the contraction to KCL (100 mmol/L). Concentration-response curves were obtained in a cumulative fashion. Several rings cut from the same artery were studied in parallel, and only one concentration-response curve was made per preparation. In quiescient rings, NG-nitro-L-arginine methyl ester (L-NAME), PD123319, and HOE 140 did not affect resting tension. Responses to acetylcholine, Ang II, and sodium nitroprusside were obtained during submaximal contraction to norepinephrine (10−6 mol/L). Incubation time was 30 min for PD123319 and HOE 140 and 15 min for L-NAME. Relaxations were expressed as the percentage of maximal relaxations induced by papaverine (3 × 10−4mol/L). In another series of experiments, the response to Ang II was assessed in aortic rings isolated from untreated SHR and WKY rats after acute AT1R blockade by incubation with losartan (10−5 mol/L) for 180 min.
Determination of NO production
Quantitative determination of NO production was carried out using a 4,5-diaminofluorescein (DAF-2) fluorescent probe as previously described.16 Briefly, aortic rings were incubated for 2 h in a dark room in Krebs solution containing DAF-2 diacetate (DAF-2DA 10 μmol/L; Alexis, San Diego, CA). Angiotensin II was administered for the last 30 min of DAF-2 incubation. Vascular rings were rapidly removed and frozen at −20°C. From 1 to 3 days after the inclusion, they were cut into sections 10 μm thick in a Jung CM-3000 cryostat (Leica, Nussloch, Germany). Sections were placed onto microscope slides without any mounting medium or cover slip. Specimens were observed under an Axiophot 2 fluorescence microscope (Zeiss, Jena, Germany) equipped with a fluorescein isothiocyanate filter (excitation 450 to 490 nm, emission 515 to 560 nm). The magnification used was ×20. Color pictures (24-bit) were obtained using a digital camera system coupled to imaging software (Spot; Diagnostic Instruments, Sterling Heights, MI) under constant exposure time, gain, and offset. To account for fluorescence decay, all images were taken in the first 30 sec of light exposure. The endothelial fluorescence intensity of four to six sections per ring was measured and expressed as fluorescence arbitrary units ranging from 0 (absolute black) to 255 (absolute green).
Isolation of total RNA and quantification of mRNA
Both AT1R and AT2R mRNA expression were measured by a semi-quantitative RT-PCR. Total RNA was isolated from the middle portion of the thoracic aorta with TRIzol solution (Invitrogen, Carlsbad, CA) and treated with RNAse-free DNAse (Promega, Madison, WI). Reverse transcription was performed in a reaction volume of 25 μL containing 2 μg of RNA with 600 U of Moloney murine leukemia virus reverse transcriptase (Promega) at 37°C for 1 h. After first-strand synthesis of RNA, 2 μL cDNA was amplified using specific primers. For amplifcation of AT1R cDNA, the sense primer was 5′-GCCCTTAACTCTTTCTGCTGAAG-3′ and the antisense primer 5′-GAAAAGCACAATCGCCATAATT-3′. For the AT2R, the sense primer was 5′-CTTTTGATAATCTCAACGCAAC-3′ and the antisense primer 5′-TGCCAACACAACAGCAGCTGCC-3′. For glyceraldehyde phosphate dehydrogenase (GAPDH), used as an internal control, the sense primer was 5′-AGACAGCCGCATCTTCTTGT-3′; and the antisense primer 5′-TGATGGCAACAATGTCCACT-3′. The amplification profile involved denaturation at 94°C for 1 min, annealing at 60°C for 50 sec, and extension at 72°C for 50 sec for 20 cycles (AT1R) and 30 cycles (AT2R). The PCR products were separated on a 1.5% agarose gel and visualized by ultraviolet light. The density of each PCR band was analyzed with a densitometer. The amount of mRNA was expressed as the ratio to GAPDH.
Drugs
Acetylcholine chloride, Ang II, L-NAME, norepinephrine, sodium nitroprusside, papaverine hydrochloride, and chemical components of the control solution were obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions of the drugs were freshly prepared every day. Losartan was a gift by Merck, Sharp & Dohme Research Laboratories (Merck & Co., Rahway, NJ). The PD 132219 was provided by Parke-Davis (Ann Arbor, MI). HOE 140 was provided by Aventis Pharma (Frankfurt, Germany). All concentrations are expressed as the final molar (mol/L) concentration in the bath solutions.
Statistical analysis
Results are expressed as mean ± SEM. In each set of experiments, n equals the number of animal used. Statistical evaluation of the data was performed by using the Student t test for paired observations or Mann-Whitney test, as appropriate. For multiple comparisons, results were evaluated by analysis of variance. A value of P < .05 was considered statistically significant.
Results
Systolic BP and heart rate
After 8 weeks, losartan reduced systolic BP (SBP) in SHR (184 ± 4 v 160 ± 4 mm Hg; P < .05), whereas it did not significantly affect SBP in WKY rats (125 ± 3 v 117 ± 3 mm Hg, P = NS). Heart rate did not differ within groups after treatment (data not shown).
Endothelium-dependent relaxations in response to acetylcholine
During contraction induced by norepinephrine (10−6 mol/L), endothelium-dependent relaxation in response to acetylcholine (10−9 to 10−5 mol/L) was reduced in SHR (P < .05 v values in WKY rats for maximal relaxation) (Fig. 1). Chronic treatment with losartan restored the response to acetylcholine (P < .05 v values in untreated SHR) (Fig. 1).
Concentration-response curves to acetylcholine in aortas isolated from sponaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats receiving or not receiving long-term treatment with losartan. Relaxations were obtained during contractions to norepinephrine (10−6 mol/L). Data are means ± SEM (n = 8 to 12). *Significant difference between SHR and the other experimental groups (P < .05).
Endothelium-independent relaxations in response to sodium nitroprusside
During contraction induced by norepinephrine, the NO donor sodium nitroprusside (10−10 to 10−5 mol/L) caused similar concentration-dependent responses in both strains (91% ± 9% v 98% ± 1% of maximal relaxation for SHR and WKY rats, respectively, n = 4). Treatment with losartan did not affect the relaxations in response to sodium nitroprusside (100% of maximal relaxation in both strains).
Vasomotor response to Ang II
As expected, treatment with losartan abolished Ang II-mediated contractions in quiescient rings isolated from both strains (10−6 to 10−4 mol/L; data not shown). Interestingly, during contractions to norepinephrine, Ang II evoked concentration-dependent relaxations only in aortic rings isolated from SHR treated with losartan for 8 weeks (P < .05 v values in untreated SHR) (Fig. 2A). By contrast, Ang II did not exert any significant effect in aortas obtained from treated WKY rats (P = NS v values in untreated animals) (Fig. 2B). Analogous experiments performed in aortas of additional SHR after acute exposure to losartan (incubation time, 180 min) failed to show Ang II-mediated relaxations (data not shown).
Concentration-response curves to angiotensin II in aortas of SHR (A) and WKY rats (B) receiving or not receiving long-term treatment with losartan. Relaxations were obtained during contractions to norepinephrine (10−6 mol/L). Data are means ± SEM (n = 8 to 12). *Significant difference between control and losartan-treated animals (P < .05). Abbreviations as in Fig. 1.
Relaxations in response to Ang II were abolished by the AT2R antagonist PD 123319 (10−5 mol/L; P < .05 v control values) (Fig. 3A). Removal of endothelium (data not shown) or inhibition of nitric oxide synthase with L-NAME (3 × 10−4 mol/L) also blunted the relaxations in response to Ang II in treated SHR (Fig. 3B). Furthermore, the relaxing effect of Ang II was significantly inhibited after incubation of treated SHR aortas with bradykinin receptor antagonist (HOE 140; 10−5mol/L) (Fig. 3C).
Effects of AT2 receptor antagonist PD123319 (A), nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (B), and bradykinin receptor antagonist HOE140 (C) on concentration-response curves to angiotensin II in aortas isolated from SHR receiving long-term treatment with losartan. Relaxations were obtained during contractions to norepinephrine (10−6 mol/L). Data are means ± SEM (n = 4 to 8). *Significant difference between control and PD123319-, L-NAME-, or HOE 140-treated rings (P < .05). Abbreviations as in Fig. 1.
Contractions to norepinephrine
Contractions due to norepinephrine (10−6 mol/L) did not differ within the four experimental groups (Table 1). In addition, treatment with L-NAME, PD 123319, and HOE 140 did not affect the response to norepinephrine (Table 1).
Effect of norepinephrine (NE) on resting tension of aortas obtained from different experimental groups
| Increase in Tension (g) | ||||
|---|---|---|---|---|
| Treatment | SHR | WKY | SHR (losartan) | WKY (losartan) |
| NE (10−6 mol/L) | 1.8 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.1 | 2.0 ± 0.2 |
| PD 123319 (10−5 mol/L) + NE | 1.7 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.2 | 2.0 ± 0.1 |
| HOE 140 (10−5 mol/L) + NE | 1.2 ± 0.2 | 2.0 ± 0.3 | 1.7 ± 0.2 | 2.0 ± 0.2 |
| L-NAME (3 × 10−4 mol/L) + NE | 1.5 ± 0.2 | 2.0 ± 0.4 | 1.5 ± 0.2 | 2.0 ± 0.4 |
| Increase in Tension (g) | ||||
|---|---|---|---|---|
| Treatment | SHR | WKY | SHR (losartan) | WKY (losartan) |
| NE (10−6 mol/L) | 1.8 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.1 | 2.0 ± 0.2 |
| PD 123319 (10−5 mol/L) + NE | 1.7 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.2 | 2.0 ± 0.1 |
| HOE 140 (10−5 mol/L) + NE | 1.2 ± 0.2 | 2.0 ± 0.3 | 1.7 ± 0.2 | 2.0 ± 0.2 |
| L-NAME (3 × 10−4 mol/L) + NE | 1.5 ± 0.2 | 2.0 ± 0.4 | 1.5 ± 0.2 | 2.0 ± 0.4 |
L-NAME = NG-nitro-L-arginine methyl ester; SHR = spontaneously hypertensive rats; WKY = Wistar-Kyoto rats.
Absolute values of tension developed by NE are shown as mean ± SEM; n = 8 for each group.
Effect of norepinephrine (NE) on resting tension of aortas obtained from different experimental groups
| Increase in Tension (g) | ||||
|---|---|---|---|---|
| Treatment | SHR | WKY | SHR (losartan) | WKY (losartan) |
| NE (10−6 mol/L) | 1.8 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.1 | 2.0 ± 0.2 |
| PD 123319 (10−5 mol/L) + NE | 1.7 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.2 | 2.0 ± 0.1 |
| HOE 140 (10−5 mol/L) + NE | 1.2 ± 0.2 | 2.0 ± 0.3 | 1.7 ± 0.2 | 2.0 ± 0.2 |
| L-NAME (3 × 10−4 mol/L) + NE | 1.5 ± 0.2 | 2.0 ± 0.4 | 1.5 ± 0.2 | 2.0 ± 0.4 |
| Increase in Tension (g) | ||||
|---|---|---|---|---|
| Treatment | SHR | WKY | SHR (losartan) | WKY (losartan) |
| NE (10−6 mol/L) | 1.8 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.1 | 2.0 ± 0.2 |
| PD 123319 (10−5 mol/L) + NE | 1.7 ± 0.1 | 2.0 ± 0.2 | 1.7 ± 0.2 | 2.0 ± 0.1 |
| HOE 140 (10−5 mol/L) + NE | 1.2 ± 0.2 | 2.0 ± 0.3 | 1.7 ± 0.2 | 2.0 ± 0.2 |
| L-NAME (3 × 10−4 mol/L) + NE | 1.5 ± 0.2 | 2.0 ± 0.4 | 1.5 ± 0.2 | 2.0 ± 0.4 |
L-NAME = NG-nitro-L-arginine methyl ester; SHR = spontaneously hypertensive rats; WKY = Wistar-Kyoto rats.
Absolute values of tension developed by NE are shown as mean ± SEM; n = 8 for each group.
Production of NO
In agreement with functional data, Ang II-induced stimulation of NO production was observed only in treated SHR aortas (P < .05 v values in control SHR and WKY rats) (Figs. 4A and 4B).
(A) Representative aortic sections from treated and untreated WKY rats and SHR showing NO production assessed by 4,5-diaminofluorescein (DAF-2) fluorescent probe after stimulation with angiotensin II (Ang II) (magnification ×20). (B) Fluorescence intensity of Ang II-induced NO production. Data are means ± SEM (n = 6 to 8). *Significant difference between treated SHR versus untreated SHR as well as WKY rats (P < .05). Abbreviations as in Figs. 1 and 3.
mRNA expression of AT1R and AT2R
Expression of AT1R and AT2R mRNA after chronic AT1R blockade in aortic tissue from SHR is shown in Figs. 5A and 5B. Treatment with losartan did not affect AT1R mRNA expression (Fig. 5A). By contrast, levels of AT2R mRNA significantly increased after AT1R blockade with losartan (P < .05 v values in control SHR) (Fig. 5B).
Effect of long-term treatment with losartan on angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) mRNA expression assessed by reverse transcription-polymerase chain reaction in aortas of spontaneously hypertensive rats (SHR). (A) Representative blots and (B) densitometric quantification of AT1R and AT2R mRNA expression normalized by GAPDH levels. Data are means ± SEM (n = 3) and expressed as fold induction. *P < .05 v values in untreated SHR.
Discussion
We demonstrate for the first time that in hypertensive rats long-term AT1R blockade unmasks AT2R-mediated relaxation in response to Ang II. Our current findings also provide evidence that downstream mechanisms responsible for Ang II-induced AT2R relaxation are represented by activation of the endothelial bradykinin/NO pathway, as observed in different experimental models.6,11
These conclusions are supported by several lines of evidence. First, exogenous Ang II caused concentration-dependent relaxations in aortic rings isolated from SHR receiving long-term treatment with the AT1R antagonist losartan. By contrast, acute exposure of SHR aortas to losartan did not reveal any relaxing effect of Ang II. In addition, Ang II did not exert any significant effect in aortas isolated from WKY rats after chronic AT1R blockade. Second, the inverted vasomotor response to Ang II is mediated via AT2R-dependent activation of the endothelial bradykinin/NO pathway. Indeed, blockade of AT2R with PD 123319 as well as bradykinin (B2) receptor antagonism with HOE 140 abolished Ang II-induced relaxations in SHR chronically receiving losartan. Hence, it is likely that bradykinin released through AT2R signaling activates endothelial B2 receptors leading to activation of endothelial nitric oxide synthase (eNOS) and NO production. Accordingly, Ang II-induced relaxation was inhibited by removal of the endothelium and inhibition of eNOS with L-NAME. Third, Ang II caused a significant stimulation of NO production only in treated SHR aortas. This latter finding provides the first direct evidence that Ang II is able to promote an AT2R-mediated NO production in aortas of hypertensive rats in the setting of chronic AT1R antagonism. Finally, marked enhancement of AT2R mRNA expression was observed in aortas isolated from treated SHR, supporting the contribution of Ang II receptor “cross-talk” during chronic treatment with losartan.
In this regard, we have previously shown that Ang II downregulates AT2R mRNA expression via AT1R binding in cultured rat aortic endothelial cells.14 Blockade of AT1R may prevent this downregulation and may increase AT2R gene promoter activity.14 On the other hand, it was recently reported that overexpression of AT2R downregulates expression of the AT1R through the bradykinin/NO pathway in rat vascular smooth-muscle cells from WKY rats.17 By contrast, in vascular smooth-muscle cells from SHR, the lack of this molecular feedback mechanism may contribute in part to exaggerated growth and vascular remodeling.17 These findings support the concept that the integrated Ang II receptor network may play a role in hypertension. Our study highlights the counter-balancing role of AT1R/AT2R on Ang II-induced vasomotor responses. It is well established that Ang II can increase BP and induce vasoconstriction by stimulating the AT1R.3 However, our understanding of the role of the AT2R in the vascular responses to Ang II and systemic BP regulation is still evolving. In this regard, it has been shown that AT2R gene disruption increases BP12,13; AT2R knock-out mice exhibit, in turn, a larger increase in BP and total peripheral vascular resistance after a low-dose infusion of Ang II.18 In contrast, mice overexpressing AT2R in vascular smooth muscle exhibited an attenuated pressor response to Ang II infusion.7 Treatment of these transgenic mice with a B2 receptor antagonist or nitric oxide synthase inhibitor restored the pressor response to Ang II.7 Thus, it is likely that AT2R-mediated vasodilation exerts an important modulatory mechanism during AT1R blockade. In animal models characterized by activation of the renin-angiotensin system, AT2R blockade substantially inhibits the BP-lowering effect of the AT1R antagonist losartan. In agreement with our present findings, AT2R stimulation has been associated with increased production of bradykinin, NO, and cGMP in different models.5–11 This effect may be relevant also for control of systemic BP.
In the present study, we have thoroughly addressed the mechanisms underlying AT2R-mediated vascular response in a hypertensive rat model. Only chronic AT1R blockade was able to reveal the AT2R-dependent activation of endothelial bradykinin/NO pathway in SHR aortas, whereas acute exposure of SHR aortas to losartan did not show any AT2R-mediated effect. Consistently, exogenous Ang II caused NO production coupled with endothelium-dependent relaxations only in aortic rings from SHR after long-term treatment with losartan.
An additional interesting observation from our studies is that endothelium-dependent response to acetylcholine was normalized only in losartan-treated SHR. Similar findings were obtained in resistance arteries isolated from patients with mild to moderate hypertension.19 The mechanisms for the beneficial effect of chronic treatment with losartan on endothelial function are unclear. Blockade of AT1R with blunted Ang II-induced oxidative stress20,21 may decrease NO breakdown. Furthermore, elevation of unbound Ang II elicited by AT1R antagonism and stimulation of AT2R may also favorably affect endothelial function.
Because AT2R gene expression is elevated during fetal life and decreases rapidly after birth,22–25 there is substantial debate concerning the relevance of AT2R-mediated effects. In adults the AT2R seems restricted to some vascular territories,22,24 although its expression may also be enhanced in pathologic conditions including hypertension, heart failure, or vascular injury.26,27 Accordingly, the AT2R-dependent NO production is observed only after long-term AT1R blockade in SHR. Our findings regarding AT2R mRNA expression in aortas isolated from SHR receiving treatment with losartan support this concept. Indeed, AT2R mRNA expression is significantly enhanced after chronic treatment with losartan only in the hypertensive strain.
Although the combination of high BP and AT1R blockade is necessary to elicit the AT2R-mediated vasorelaxation in response to Ang II, it is difficult to draw any conclusion about the relative role played by either AT1R blockade or the hypertensive state in modulating the observed AT2R expression and function. In agreement with our results, previous reports demonstrated that AT2R stimulation increases aortic cyclic GMP in stroke-prone SHR, whereas it did not contribute to the cyclic GMP production in the vascular wall of normotensive WKY rats.28
In view of the extensive use of AT1R blockers in cardiovascular disease, a more thorough characterization of the AT2R-mediated vasomotor effects is important. Our study highlights the complex mechanisms underlying the BP-lowering and vasculoprotective effects of these compounds.
