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(Circulation. 2005;111:1006-1011.)
© 2005 American Heart Association, Inc.

Hypertension

High Blood Pressure Reduction Reverses Angiotensin II Type 2 Receptor–Mediated Vasoconstriction Into Vasodilation in Spontaneously Hypertensive Rats

Dong You, BSc; Laurent Loufrani, PhD; Celine Baron, BSc; Bernard I. Levy, MD, PhD; Robert E. Widdop, PhD; Daniel Henrion, PharmD, PhD

From INSERM Unit 541 (D.Y.) and Department of Physiology (B.I.L.), AP-HP-Hôpital Lariboisière, Paris, France; UMR-CNRS 6188, University of Angers, Angers, France (L.L., C.B., D.H.); and Department of Pharmacology, Monash University, Melbourne, Australia (R.E.W.).

Correspondence to D. Henrion, PharmD, PhD, Laboratoire de Physiologie, UMR-CNRS 6188, Faculté de Médecine, 49045 Angers, France. E-mail daniel.henrion{at}med.univ-angers.fr

Received May 20, 2004; revision received September 28, 2004; accepted November 10, 2004.

	Abstract

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Background— We have previously shown that angiotensin II type 2 receptor (AT₂R) stimulation causes endothelium-dependent vasodilation that does not desensitize after chronic angiotensin II type 1 receptor (AT₁R) blockade, suggesting a role for AT₂R in antihypertensive treatment.

Methods and Results— We recorded mean arterial pressure (MAP) and investigated AT₂R by Western blot analysis, immunohistochemistry, and function in isolated mesenteric resistance arteries (205 µm in diameter) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) receiving the following for 4 weeks in drinking water: placebo, AT₁R blockade (candesartan; 2 mg/kg per day), ACE inhibitor (perindopril; 3 mg/kg per day), nonselective vasodilator (hydralazine; 16 or 24 mg/kg per day), or candesartan plus hydralazine (16 mg/kg per day). In precontracted isolated arteries, AT₂R stimulation (angiotensin II in the presence of candesartan) caused vasodilation in WKY rats (MAP=118 mm Hg) and vasoconstriction in SHR (MAP=183 mm Hg). In SHR treated with candesartan (MAP=146 mm Hg) or hydralazine (16 mg/kg per day; MAP=145 mm Hg), AT₂R-induced contraction was reduced by 50%. In SHR treated with perindopril (MAP=125 mm Hg), AT₂R stimulation induced vasodilation. In SHR treated with hydralazine (24 mg/kg per day; MAP=105 mm Hg) and in SHR treated with hydralazine (16 mg/kg per day) plus candesartan (MAP=102 mm Hg), an AT₂R-mediated vasodilation was restored. Immunochemistry and Western blot analysis showed that AT₂R expression, lower in SHR than in WKY rats, was restored to normal levels by treatments reducing arterial pressure in SHR.

Conclusions— Our results suggest that in resistance arteries of SHR, (1) AT₂R is downregulated by hypertension, and (2) specific and nonspecific antihypertensive treatments restore AT₂R expression and vasodilator functions.

Key Words: angiotensin • receptors • vasculature • vasodilation • hypertension

	Introduction

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Angiotensin II (Ang II), the key effector of the renin-angiotensin-aldosterone system, has an important role in the control of blood pressure and blood volume. Ang II activates at least 2 receptor types^1,2: the Ang II type 1 receptors (AT₁R) and the Ang II type 2 receptors (AT₂R).^3–5 Stimulation of AT₂R undoubtedly induces relaxation in several vascular territories.^6,7 In most blood vessels, AT₂R-dependent relaxation is associated with activation of the bradykinin or NO/cGMP pathway.^8–11 In vitro, the vasodilator role of AT₂R is supported by evidence based on enhanced Ang II–mediated vasoconstriction in the presence of AT₂R blockade or in _AT2R-knockout mice.^12–14 AT₂R mRNA and protein expression were demonstrated in resistance arteries from normotensive rats.^15,16 We have previously shown in mesenteric resistance arteries from Wistar-Kyoto (WKY) rats that AT₂R is involved in NO-dependent flow-mediated dilation

See p 956

,¹⁶ whereas in spontaneously hypertensive rats (SHR), flow (shear stress) stimulation of the endothelium is associated with an AT₁R and endothelin-1 type A receptor activation, thus counteracting endothelium-dependent dilation.¹⁷ Furthermore, acute administration of an AT₂R inhibitor reversed both the acute antihypertensive effects and elevated level of bradykinin, NO, and cGMP in renal interstitial fluid caused by AT₁R blockade in renal wrap and salt-restricted rats.^18,19 Finally, we have recently shown the reproducibility of the vasodilator effect of AT₂R stimulation under acute and chronic AT₁R blockade,²⁰ further supporting the assumption that AT₂R stimulation might play a role in the antihypertensive effect of AT₁R-blocking drugs. Thus, we speculate that AT₂R stimulation, and hence vasodilation, might play a role in antihypertensive treatments, especially when AT₁R-blocking agents are used. Indeed, AT₁R antagonists induce an important rise in circulating Ang II. AT₂R might then be chronically overstimulated, participating in the maintenance of vasodilation, particularly because this effect was not easily desensitized.²⁰

Thus, in the present study we evaluated the vasomotor role of AT₂R in resistance arteries isolated from SHR (hypertensive conditions) and after various chronic antihypertensive treatments, including ACE inhibition, AT₁R blockade or a nonselective treatment, and AT₂R expression and immunolocation. We hypothesized that AT₂R-mediated dilation, which was impaired in untreated SHR, would be reestablished in mesenteric arteries taken from treated SHR in parallel with reductions in blood pressure.

	Methods

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Animal Model
Male WKY rats and SHR (aged 7 to 8 weeks) were separated into 8 groups receiving the following for 4 weeks in drinking water: WKY rats, placebo or AT₁R antagonist (candesartan cilexetil; 2 mg/kg per day); SHR, placebo, ACE inhibitor (perindopril; 3 mg/kg per day), candesartan (2 mg/kg per day), nonselective antihypertensive drug (hydralazine; 2 groups: 16 or 24 mg/kg per day), or candesartan plus hydralazine (16 mg/kg per day).

The protocol used was in accordance with the European Community standards on the care and use of laboratory animals (authorization No. 00577).

Mean Arterial Pressure Measurement
After 4 weeks of treatment, rats were anesthetized with sodium pentobarbital (50 mg/kg IP). Mean arterial pressure (MAP) was measured in the right carotid artery with a catheter connected to a GOULD transducer and an analog-digital signal recording system (Biopac).^16,17

Isolated Mesenteric Artery
A 3- to 4-mm-long segment of mesenteric artery (205±11 µm, internal diameter measured at 75 mm Hg in the absence of tone) was dissected, cannulated at both ends, and mounted in a video-monitored perfusion system,²¹ as we have previously described.^16,17,20 Briefly, arteries were bathed in a physiological salt solution (PSS) maintained at 37°C, pH 7.4. The PO₂ was 160 mm Hg and the PCO₂ 37 mm Hg.^16,17,20 The artery was superfused (4 mL/min), and flow through the vessel was maintained at a rate of 100 µL/min, with intraluminal pressure set at 75 mm Hg.²⁰ Arterial diameter was measured (Living Systems Instrumentations) and recorded continuously (Biopac). Vessels were allowed to stabilize for at least 30 minutes before drugs were added to the PSS superfusion. The integrity of the endothelium was assessed by testing the relaxing effect of acetylcholine (1 µmol/L) after precontraction with phenylephrine (1 µmol/L). The arteries were then exposed to candesartan (100 nmol/L) for at least 30 minutes before exposure to Ang II while they were precontracted with phenylephrine (1 µmol/L) and serotonin (0.1 µmol/L) to induce a stable reduction in diameter. When this response had reached a plateau, a concentration-response curve to Ang II at 0.1 to 100 nmol/L or Ang II at 100 nmol/L was performed, and changes in diameter were measured. Acetylcholine (1 µmol/L) was used to completely dilate the vessels. Eight rats were used in each group.

In separate series of experiments (n=5 per group), AT₂R stimulation was repeated before and after application of one of the following drugs: the cyclooxygenase inhibitor indomethacin (10 µmol/L), the thromboxane A₂-PGH₂ receptor blocker SQ29548 (10 µmol/L), the bradykinin B₂ receptor blocker HOE140 (0.1 µmol/L), and the endothelin receptor blocker bosentan (10 µmol/L).

Stimulation of AT₂R was also performed before and after endothelium removal (5 seconds of air perfusion; n=4 rats per group) in WKY rats, untreated SHR, and SHR treated with candesartan plus hydralazine (16 mg/kg per day).

Western Blot Analysis of AT₂R
Western blot analysis of AT₂R was performed in mesenteric resistance arteries of WKY rats and SHR (n=8 per group). Mesenteric arteries were also isolated from SHR treated with hydralazine (24 mg/kg per day for 18, 23, or 48 days; n=5 per group) or with candesartan plus hydralazine (16 mg/kg per day; n=5 per group). Mesenteric arteries were homogenized with a lysis buffer (1% sodium dodecyl sulfate, 10 mmol/L Tris-HCl [pH 7.4], 1 mmol/L sodium orthovanadate, 2.5 mg/L leupeptin, and 5 mg/L aprotinin). Extracts were incubated at 25°C for 30 minutes and then centrifuged (1000g, 15 minutes, 14°C). Protein concentration was determined with the use of the Micro BCA Protein Assay Kit (Pierce). After denaturation at 100°C for 5 minutes, equal amounts of proteins (15 µg) were loaded on a 9% polyacrylamide gel and transferred to nitrocellulose membranes for 12 hours (40 V, 4°C). Membranes were blocked with 10% BSA in TBST (20 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, and 0.1% Tween-20) for 1 hour and were then incubated with AT₂R rabbit polyclonal antibody (dilution 1:100, Santa Cruz) in washing solution at room temperature for 20 hours. The membranes were then washed and incubated with the anti-rabbit horseradish peroxidase antibody (dilution 1:5000; Amersham Pharmacia Biotech) for 1 hour at room temperature. After 3 washes with TBST, immunocomplexes were detected by chemiluminescent reaction (ECL kit; Amersham Pharmacia Biotech) with a computer-based imaging system (Fuji LAS 1000 Plus; Fuji Medical Systems). Quantification was performed by densitometric analysis.

Immunofluorescence Analysis of AT₂R
Segments of mesenteric resistance arteries (n=6 rats per group) were mounted in embedding medium (Miles, Inc), frozen in isopentane precooled in liquid nitrogen, and stored at –80°C on transverse cross sections 7 µm thick. Sections were incubated with candesartan (30 minutes, 10 nmol/L, 25°C), then with fluorescent Ang II (FITC-bound Ang II, 30 minutes, 10 pmol/L, 25°C; Molecular Probes). Fluorescence staining was visualized by confocal microscopy (Biorad MRC-600). Control experiments were performed after incubation with nonfluorescent Ang II. Image analysis was performed with the use of Histolab (Microvision). Briefly, pixel quantification was performed after the media and the endothelial layer were separated. Data are given as percentage of control (with fluorescence in WKY rats taken as 100%).

Drugs
Candesartan cilexetil was kindly provided by AstraZeneca (Sweden). Other products were purchased from Sigma.

Statistical Analysis
Results are expressed as mean±SEM. The significance of the different treatments was determined by ANOVA or 2-tailed Student paired t test. Probability values <0.05 were considered significant. Number of rats was used for the analysis.

	Results

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MAP and AT₂R-Mediated Dilation in Isolated Arteries
Figure 1 shows typical recordings obtained with mesenteric arteries isolated from WKY rats (Figure 1A) and SHR (Figure 1B). MAP was 118±8 mm Hg (n=8) in WKY rats and 183±11 mm Hg (n=8) in SHR. Isolated arteries were first incubated with candesartan (100 nmol/L, 30 minutes) and precontracted with phenylephrine (54±4 µm diameter decrease). Addition of Ang II (100 nmol/L) induced a significant dilation (24±3 µm diameter increase) in control WKY rats. By contrast, in SHR the stimulation of AT₂R led to a significant contraction (8±3 µm diameter decrease; Figure 1B). In both WKY rats and SHR, the arteries were able to fully dilate when acetylcholine was added after Ang II. Diameter changes in response to AT₂R stimulation (dilation in WKY rats or contraction in SHR) were suppressed by the AT₂R antagonist PD123319 (1 µmol/L). In the presence of candesartan and PD123319, Ang II produced no significant change in diameter (2±3 µm, n=5 in WKY rats and –1±2 µm, n=4 in SHR).

View larger version (31K): [in this window] [in a new window]   Figure 1. Typical recordings obtained in mesenteric arteries isolated from WKY rats (A) and SHR (B) and perfused under a pressure of 75 mm Hg, a flow of 100 µL/min, and in the presence of candesartan (100 nmol/L). After precontraction with phenylephrine (PE), Ang II (100 nmol/L) was added to the bath containing the artery. Finally, acetylcholine (ACh) (1 µmol/L) was added to fully relax the artery (n=8 per group). C, Concentration-response curves to Ang II obtained in the conditions described above.

The contraction induced by Ang II (100 nmol/L) in arteries isolated from SHR was significantly reduced by indomethacin (10 µmol/L; 6±2 versus 12±3 µm reduction in diameter; n=5), SQ29548 (10 µmol/L; 6±3 versus 14±3 µm reduction in diameter; n=5) and by bosentan (10 µmol/L; 7±3 versus 16±3 µm reduction in diameter; n=5). The combination of indomethacin (10 µmol/L) and bosentan (10 µmol/L) suppressed Ang II–induced contraction in SHR (2±3 versus 14±3 µm reduction in diameter; n=5). The bradykinin B₂ receptor blocker HOE140 (0.1 µmol/L) did not significantly affect Ang II–induced contraction in SHR (13±3 versus 15±4 µm reduction in diameter). In WKY rats, Ang II–induced dilation was significantly reduced by N^G-nitro-L-arginine methyl ester (L-NAME) (4±2 versus 24±4 µm) and by HOE140 (6±3 versus 26±5 µm).

The stimulation of AT₂R produced a concentration-dependent dilation in arteries isolated from WKY rats and a concentration-dependent contraction in arteries isolated from SHR (Figure 1C; n=8 per group).

Concentration-dependent stimulation of AT₂R (Ang II 0.01 to 100 nmol/L) was repeated in arteries isolated from WKY rats and SHR submitted to various treatments. Maximal responses to AT₂R stimulation and MAP determined in the different groups are shown in Figure 2.

View larger version (22K): [in this window] [in a new window]   Figure 2. A, Ang II (100 nmol/L)–mediated dilation (diameter increase) or contraction (diameter decrease) in the presence of candesartan (100 nmol/L), obtained in isolated mesenteric arteries of normotensive rats (WKY) or SHR perfused in an arteriograph under a pressure of 75 mm Hg and a flow of 100 µL/min. Rats were treated for 4 weeks with candesartan (TCV; 2 mg/kg per day), perindopril (PER; 3 mg/kg per day), hydralazine (hydra; 16 or 24 mg/kg per day), or candesartan plus hydralazine (TCV 2 mg/kg per day plus hydra 16 mg/kg per day). B, MAP measured in the groups of rats described above. Data are mean±SEM (n=8 per group). C, Relationship between MAP and AT2R-mediated diameter changes in resistance arteries (from data shown in A and B). Ctr indicates control; Cand, candesartan. *P<0.05 vs control WKY; #P<0.05 vs control SHR.

There was no significant difference in MAP and AT₂R-mediated dilation between control WKY rats (118±8 mm Hg; 24±3 µm diameter increase; n=8) and WKY rats treated with candesartan (108±9 mm Hg; 19±5 µm diameter increase; n=4).

In SHR, candesartan partly depressed MAP (146±8 mm Hg, n=4 versus 183±11 mm Hg, n=8; P<0.01 versus SHR and P<0.01 versus WKY rats) and tended to reduce AT₂R-mediated contraction, although this did not reach significance (8±3 versus 3±2 µm diameter decrease).

In SHR, treatment with perindopril reduced MAP (n=4; 125±6 mm Hg; P<0.01), and AT₂R stimulation induced a significant vasodilation (6±2 µm diameter increase) that was significantly lower than AT₂-induced dilation in WKY rats.

In SHR treated with hydralazine (16 mg/kg per day; n=4), MAP decreased to a level that was still higher than in WKY rats (145±11 mm Hg; n=6; P<0.01 versus SHR and P<0.05 versus WKY rats), and stimulation of AT₂R induced vasoconstriction (3±1 µm diameter decrease; n=6).

In SHR treated with a higher dose of hydralazine (24 mg/kg per day; n=4), MAP was reduced to a normal value (105±10 mm Hg; P=NS versus WKY rats), and AT₂R induced a significant vasodilation (27±7 µm diameter increase; P=NS versus WKY rats).

In SHR treated with candesartan plus hydralazine (16 mg/kg per day), MAP was reduced to a normal level (n=4; 102±9 mm Hg; P=NS versus WKY rats), and AT₂R induced a significant vasodilation (22±5 µm diameter increase; P=NS versus WKY rats).

Endothelium removal did not affect AT₂R-dependent contraction in untreated SHR (12±3 µm with endothelium versus 14±3 µm contraction without endothelium; n=4). On the other hand, AT₂R-dependent dilation was abolished by endothelium removal in both untreated WKY rats (24±4 versus 3±2 µm dilation; n=4) and SHR treated with candesartan plus hydralazine (16 mg/kg per day; 26±4 versus 4±2 µm increase in diameter; n=4).

Western Blot Analysis of AT₂R
In resistance arteries isolated from untreated WKY (n=8) rats and untreated SHR (n=8), as well as from SHR treated with hydralazine alone (24 mg/kg per day; n=5) or hydralazine (16 mg/kg per day; n=5) combined with candesartan, AT₂R expression was quantified. Western blot analysis showed that AT₂R was significantly less expressed in SHR than in WKY rats (46%) in isolated mesenteric resistance arteries (Figure 3). Hydralazine (24 mg/kg per day) gradually raised AT₂R expression in SHR treated for 18, 23, and 48 days. After 48 days, AT₂R expression was significantly higher than in SHR, but it remained significantly lower than in WKY rats (70%; Figure 3). However, after 44 days of treatment, candesartan plus hydralazine (16 mg/kg per day) restored AT₂R expression in SHR to a level equivalent to that found in WKY rats (96.7% versus 100%; Figure 3).

View larger version (62K): [in this window] [in a new window]   Figure 3. Western blot analysis of AT2R in mesenteric resistance arteries of normotensive rats (WKY; white bar; n=8) or SHR (black bar; n=8). One group of SHR was treated with hydralazine (Hydra; 24 mg/kg per day) for 18, 23, or 48 days (n=5 per group). Another group of SHR was treated with candesartan plus hydralazine (TCV plus Hydra 16 mg/kg per day; n=5 per group). Representative blots are shown below. *P<0.05 vs control WKY; #P<0.05 vs control SHR.

Immunohistology Analysis of AT₂R
In WKY rats, immunofluorescence analysis of mesenteric resistance arteries, with the use of confocal microscopy, indicated that AT₂R was present in the endothelium, in the smooth muscle, and in the adventitia. In the endothelium, AT₂R immunofluorescence was lower in SHR than in WKY rats (5±3% of WKY; P=NS; n=6 per group). In the media a significant fluorescence could be detected, although it was lower than in WKY rats (32±6%; P<0.01; n=6). In SHR treated with candesartan plus hydralazine (16 mg/kg per day), immunofluorescence of AT₂R was restored to a level equivalent to that found in WKY rats (compared with WKY rats: 88±11% in the endothelium and 81±18% in the media) (Figure 4).

View larger version (70K): [in this window] [in a new window]   Figure 4. Confocal immunohistological analysis of AT2R in mesenteric resistance arteries of normotensive (WKY) rats, SHR, or SHR treated with candesartan (CV; 2 mg/kg per day) and hydralazine (Hydra; 16 mg/kg per day). Each image is representative of 4 different arteries (6 rats).

	Discussion

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The present study demonstrates that AT₂R stimulation induced a vasoconstriction in untreated SHR resistance arteries associated with a decrease in AT₂R expression. Specific or nonspecific antihypertensive treatments restored AT₂R expression and its vasodilator function when the decrease in pressure was sufficient.

Recent evidence suggests that AT₂R stimulation causes vasodilation in small resistance arteries in normotensive rats.^{6,7,11,16,17,20,22} This vasodilation may play an important role in the regulation of arterial blood pressure by increasing the diameter of resistance arteries. Vasodilation induced by AT₂R stimulation has been described in several vascular territories and is usually associated with NO production by endothelial cells and cGMP production by smooth muscle cells.²³ In some but not all arteries investigated, bradykinin B₂ receptor activation is involved in AT₂R-dependent dilation.^9–11,22 Importantly, AT₂R-mediated dilation does not desensitize, in contrast to AT₁R-dependent contraction, supporting the assumption that AT₂R dilation might have a role in the treatment of hypertension.^20,24 Furthermore, in agreement with previous studies,^21,25 AT₂R was found in resistance arteries by Western blot and RT-PCR analysis. With the use of immunofluorescence, AT₂R was localized in endothelial and smooth muscle cells, as in previous studies in rat mesenteric arteries¹⁶ and skeletal muscle arterioles.²⁶

We found that AT₂R stimulation in SHR induced a vasoconstriction, in agreement with previous studies showing that in young hypertensive rats Ang II–induced contraction was decreased by AT₂R blockade.²⁵ Interestingly, this effect involved stimulation of endothelin-1 and probably thromboxane A₂. We found that AT₂R expression was lower in SHR resistance arteries than in WKY rats. However, a decreased expression cannot readily explain a reversal of dilation into contraction. The mechanism of this reversal remains to be discovered but may involve a switch in signaling from constrictor to dilator mechanisms due to increased endothelial AT₂R expression. Indeed, our immunohistological analysis of AT₂R in mesenteric arteries from SHR showed undetectable AT₂R labeling in the endothelium that was reestablished with antihypertensive treatment that normalized blood pressure. In addition, AT₂R-dependent contraction in SHR is not affected by endothelium removal, whereas AT₂R-dependent dilation is abolished in the absence of endothelium. Thus, the difference in the type of response might reflect a change in AT₂R expression between the endothelium and the smooth muscle. Nevertheless, because of the small size of the resistance arteries, AT₂R expression and mRNA level were not significantly decreased by endothelium removal in either SHR or WKY rats (D. Henrion, unpublished data). In addition, cultured endothelial cells rapidly lose AT₂R phenotype, thus preventing a study of AT₂R expression in cultured endothelial cells from WKY rats or SHR.

Stimulation of the NO-cGMP pathway by AT₂R has been shown initially in aortic cells.²⁷ In the dog coronary circulation, NO production by Ang II is activated through both AT₁R and AT₂R stimulation.²⁸ Other evidence suggests that AT₁R and AT₂R may not always oppose each other, at least with regard to NO production.²³ In the mesenteric circulation, we found that AT₂R-dependent flow-mediated dilation requires NO production and that exogenous stimulation of AT₂R in the same arteries produces dilation.¹⁶

In WKY rats, candesartan did not significantly change MAP, and AT₂R-mediated dilation was preserved, as we have previously reported.²⁰ By contrast, candesartan partly decreased MAP and AT₂R-mediated vasoconstriction in SHR. Similarly, low-dose hydralazine, also partly reducing MAP, inhibited AT₂R-mediated vasoconstriction. Obviously, these treatments were not able to normalize MAP and to restore AT₂R vasodilator function. Indeed, it was only when MAP was sufficiently reduced in SHR that AT₂R-mediated vasodilation was observed. First, with the use of an ACE inhibitor, MAP was decreased to a level similar to that observed in WKY rats. In this case, stimulation of AT₂R induced vasodilation. A further decrease in MAP, with the use of a higher dose of hydralazine or candesartan plus hydralazine, was also associated with a vasodilator effect of AT₂R stimulation.

Interestingly, the different groups studied allowed a correlation to be drawn between MAP and the type and amplitude of the response to AT₂R stimulation (Figure 2). Thus, from high to low MAP, AT₂R stimulation moved progressively from contraction to dilation. In addition, this effect was associated with a different AT₂R expression. In SHR, AT₂R expression was low in the wall of mesenteric arteries compared with WKY. After MAP reduction in SHR, AT₂R expression was restored to the level of WKY rats. However, it is difficult to determine whether AT₂R-mediated dilation is a cause or a consequence of the reduction in blood pressure. In the combined candesartan/hydralazine group of SHR, the time course for AT₂R expression, assessed by Western blots, was in parallel with the restoration of maximal AT₂R-mediated dilation and normotension, which may indicate a primary role for AT₂R. On the other hand, equivalent reductions in MAP caused by hydralazine were associated with maximal AT₂R-mediated dilation despite suppressed AT₂R expression. These discrepancies in AT₂R abundance between treatments may reflect distinct AT₂R locations within the vasculature. Indeed, more precise immunohistological analysis demonstrated that AT₂R expression was located in the endothelial and smooth muscle cells in WKY rats, whereas in SHR AT₂R was not detectable in the endothelium. On the other hand, in SHR treated with candesartan plus hydralazine, MAP was restored, AT₂R stimulation induced vasodilation, and AT₂R expression was equivalent to that found in WKY rats. In this group, immunohistology of AT₂R showed the presence of the receptor in both endothelia and smooth muscle cells. Thus, we can speculate that the presence or absence of AT₂R on endothelial cells has a key role in determining the type of response, at least in part because the inhibition of the vasodilation in WKY rats by L-NAME does not uncover a vasoconstriction due to receptors located on the muscle, as revealed by immunohistology. We can also assume that MAP per se is the effector determining the type of response induced by AT₂R stimulation (Figure 2C).

In conclusion, in resistance arteries of SHR, (1) AT₂R is downregulated by hypertension, and (2) specific and nonspecific antihypertensive treatments restore AT₂R expression and vasodilator functions. Whether or not this AT₂R plasticity directly contributes to the blood pressure reduction, this AT₂R dilator mechanism is likely to contribute to the maintenance of a vasodilator state during chronic treatment.

	Acknowledgments

 
This work was supported in part by a grant from the French Foundation for Medical research (FRM, Paris, France).

	References

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