Vascular Pharmacology 54 (2011) 29–35

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Vascular Pharmacology

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Lack of heterologous receptor desensitization induced by angiotensin II type 1receptor activation in isolated normal rat thoracic aorta

Teresa Pérez, Ruth M. López, Pedro López, Carlos Castillo, Enrique F. Castillo ⁎Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina del Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n, Col. Casco de Santo Tomás, CP 11340,México, D.F., México

Abbreviations: RAS, renin–angiotensin system;angiotensin II type 1; AT2, angiotensin II type 2; GPCRBK, bradykinin; NO, nitric oxide; cGMP, cyclic guanosinnitro-L-arginine methyl ester.⁎ Corresponding author. Tel.: +52 5 729 6300x62821

E-mail address: ecastilloh@ipn.mx (E.F. Castillo).

1537-1891/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.vph.2010.11.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 September 2010Received in revised form 8 November 2010Accepted 23 November 2010

Keywords:Angiotensin IIAT receptorsDesensitizationRat aorta

We tested whether heterologous receptor desensitization induced by activation of AT1 receptors may explainthe purported relaxation produced by angiotensin II in normal rat aorta. Also, the role for AT2 receptors in thepromotion of vasodilation was studied. In endothelium-intact and endothelium-denuded aortic rings,angiotensin II elicited biphasic contractions, which were significantly depressed when repeated in each tissue.Angiotensin II produced biphasic responses on phenylephrine preconstricted endothelium-intact andendothelium-denuded tissues, without reducing precontractile tone. These responses were abolished in thepresence of the AT1 receptor antagonist losartan, but no relaxing responses to angiotensin II were uncovered.PD123319 did not influence angiotensin II responses in endothelium-intact tissues precontracted withphenylephrine; thus, under AT2 receptors blockade the contractile effects of angiotensin II were notoverexposed. In conclusion, angiotensin II-induced biphasic responses can be attributed to AT1 receptorsactivation and rapid desensitization with time. Desensitization proved to be homologous in nature, sinceprecontractile tone induced by phenylephrine was not depressed by angiotensin II (i.e., angiotensin II did notinduce heterologous α1-adrenergic receptors desensitization). We found no functional evidence of theparticipation of AT2 receptors in angiotensin II elicited biphasic contractions. Angiotensin II does not exertrelaxant effects in normal rat aorta.

ANG II, angiotensin II; AT1,, G-protein coupled receptors;monophosphate; L-NAME, NG-

; fax: +52 5 341 2726.

l rights reserved.

© 2010 Elsevier Inc. All rights reserved.

1. Introduction

The renin–angiotensin system (RAS) plays a critical role in thecontrol of cardiovascular and renal functions (Stroth and Unger,1999). Angiotensin II (ANG II) as the main active peptide of the RASacts at two G-protein coupled receptors (GPCR), ANG II type 1 (AT1)and ANG II type 2 (AT2) receptors; which have been defined on thebasis of their differential pharmacological and biochemical properties(Inagami et al., 1994; Matsubara and Inada, 1998). At present time, ithas been established beyond doubt that ANG II activation of AT1receptors induces vascular smoothmuscle contraction, which leads toincreased blood pressure. On the other hand, during the last years,evidence has accumulated that documents a physiological role for AT2receptors in the promotion of vasodilation in resistance (Baranov andArmstead, 2005; Batenburg et al., 2004; Hannan et al., 2003; Widdopet al., 2002) and capacitance vessels (Arun et al., 2004; Fukada et al.,2005; Tsutsumi et al., 1999). Since ANG II binds with similar affinity to

AT1 and AT2 receptors (Carey and Siragy, 2003; Nouet and Nahmias,2000) the corollary is that the vasoconstriction mediated by AT1receptors activationmay be physiologically opposed by AT2 receptors-induced vasodilation.

Considering in particular large-conductance vessels, currentinformation suggests that AT1 and AT2 receptors may be functionallyexpressed in rat aorta. Lately, it has been shown that AT2 receptorslocated in smooth muscle of normal rat aortic rings may mediatevasorelaxation via stimulation of the NO/cGMP pathway, vasodilatorcyclooxygenase products, and voltage-dependent and Ca2+-activatedlarge-conductance K+ channels (Fukada et al., 2005). Also, anothergroup of researchers (Arun et al., 2004) reported that ANG II produceda concentration-dependent relaxation in endothelium-intact andendothelium-denuded rat thoracic aorta in the presence, but not inthe absence, of AT1 selective antagonists (losartan or valsartan). Inaddition, PD 123319 blocked the relaxation response to Ang II,suggesting that the response is mediated through AT2 receptors (Arunet al., 2004). Importantly, there appears to be an up-regulation of AT2receptors in rat thoracic aorta under conditions associated withvascular tissue damage, such as diabetes and hypertension (Arunet al., 2004; Cosentino et al., 2005). On the other hand, it is well knownthat the contractile responses induced by ANG II in rat aorta manifestpronounced desensitization (Kuttan and Sim, 1993; Li et al., 1995; Simand Kuttan, 1992). Several studies have shown that desensitization

30 T. Pérez et al. / Vascular Pharmacology 54 (2011) 29–35

(tachyphylaxis) to ANG II is associated with changes in both theaffinity of the ligand for the AT1 receptors and the coupling efficiencyof the receptor system (Kuttan and Sim, 1993; Li et al., 1995; Sim andKuttan, 1992). Desensitization to agonists, in particular to G-proteincoupled receptor (GPCR)-agonists, is an extensively studied cellularprocess (Kelly et al., 2008; Kohout and Lefkowitz, 2003). Desensiti-zation to GPCR-agonists can be homologous or heterologous innature; homologous desensitization refers to the loss of responsesolely to agonists that act at a particular GPCR type, whereasheterologous desensitization refers to a broader effect involving thesimultaneous loss of agonist responsiveness at multiple GPCR types(Kelly et al., 2008). Accordingly, vascular tissues precontracted with aGPCR-agonist may possibly show heterologous desensitization toanother GPCR-agonist tested during the precontractile plateau.

To our knowledge, it has not been adequately studied if the relaxingresponse obtainedwith ANG II in precontracted normal rat aorta is theresult of activating AT2 receptors, exclusively (Arun et al., 2004;Fukada et al., 2005); or whether heterologous desensitization is anevent responsible, at least in part, of the relaxant process (i.e., thevasorelaxation induced by ANG IImight also reflect rapid heterologousdesensitization of the GPCR that mediates the precontractile tone).Hence, the purpose of the present studywas to investigate the relativeparticipation of AT2 receptors and heterologous receptor desensitiza-tion in the relaxation produced by ANG II in normal rat aorta.

2. Materials and methods

The experimental protocol was approved by the Animal Care andUse Committee of our Institution, and complied with the NationalHealth andMedical Research Council of México guidelines. MaleWistarrats (250–300 g body weight; 10–12 weeks old) were housed undercontrolled conditions (22±2 °C, 60% humidity and artificial light from06:00 to 18:00 h). Normal chow and tap water were given ad libitum.

2.1. Tissue preparation and measurement of tension

Rats were anesthetized with pentobarbital sodium (50 mg/Kg, i.p.)and exsanguinated. The thoracic aortae were cleaned of fat andconnective tissue, and cut into ring segments (4–5 mm in length). Insome preparations, the endothelium was damaged by gently abradingthe intimal surface of aortic rings with the tip of small forceps. Aorticrings were mounted in 10-ml tissue baths filled with physiologicalsaline solution containing (mM): NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO4

1.2, CaCl2 2.5, NaHCO3 25, EDTA 0.03, and dextrose 11.7. The mediumwas maintained at 37 °C, pH 7.4, and gassed continuously with 95% O2

and 5%CO2. Each tissuewas placed under an initial resting tension of 2 gweight and equilibrated for 60 min prior to the execution ofexperimental protocols. Contractions were measured isometricallyand recorded on a computer with the AcqKnowledge software(MP100WSW, Biopac Systems, Inc.; Santa Barbara, CA, USA). Tissueswere primed by the addition of 1×10−6 M phenylephrine to the organbath. After a steady-state contraction was achieved, bath contents werereplacedwith drug-free buffer several times. Tissues were then allowedto reach baseline tensionand theprimingprocedurewas repeated twicemore before the execution of experimental protocols. Functionalendothelium was checked by the presence of at least 80% relaxation inresponse to acetylcholine (1×10−6 M) after pre-constricting the tissueswith phenylephrine (1×10−6 M). Otherwise, successful endothelialdenudation was confirmed by the presence of small (lower than 10%)relaxations, or complete absence of relaxations, to acetylcholine.

2.2. Effects of ANG II on endothelium-intact and endothelium-denudedaortic rings

After equilibration, cumulative concentration–response curves forANG II (1×10−10 to 1×10−5 M) were constructed in endothelium-

intact and endothelium-denuded aortic ring preparations. Next, therings were rinsed four times and re-equilibrated for 1 h. A secondseries of concentration–response curves for ANG II was then built.Cumulative concentration–response curves to ANG II were alsoconstructed in endothelium-intact aortic rings after 30 min of in-cubation with the NO synthase inhibitor, NG-nitro-L-arginine methylester (L-NAME, 100 μM).

In separate experiments, the temporal course of a single low orhigh concentration of ANG II (either 1×10−8 or 1×10−5 M) wastested in endothelium-intact and endothelium-denuded tissues.Afterwards, the rings were rinsed four times and re-equilibrated for1 h, and the same concentration of ANG II was administered again.

2.3. Cumulative concentration–response curves to ANG II underconditions of active tone induced by the α1-adrenergic agonistphenylephrine

After the equilibrationperiod, endothelium-intact andendothelium-denudedaortic ringswere constrictedwith phenylephrine (1×10−7 M)and thecontractionswere allowed to stabilize. Subsequently, ANG IIwasapplied cumulatively to obtained concentration–response curves. Theeffects of ANG II were tested in the presence or absence of selective AT1or AT2 receptor blockers, losartan (100 nM) and PD123319 (1 μM),respectively. Each antagonist was applied 30 min before cumulativeadministration of ANG II. Only one cumulative concentration–responsecurve for ANG II was constructed in each tissue.

In other selective group of experiments, a sole concentration ofANG II (1×10−5 M) was tested—throughout 1 h—in endothelium-intact and endothelium-denuded aortic rings precontracted withphenylephrine (1×10−7 M).

In these sets of experiments with ANG II, vehicle (assay buffer)-treated rings served as temporal controls.

2.4. Effects of angiotensin receptors antagonists on the concentration–response curves to ANG II

Endothelium-intact and endothelium-denuded aortic rings weretreated with different concentrations of losartan (10, 30, or 100 nM),PD123319 (0.1, 1, or 10 μM), or vehicle for 30 min. (Only one con-centrationof antagonistwas tested in each tissue.) Then, concentration–response curves were established by exposing the aortic rings toincreasing concentrations of ANG II until maximal contractile responseswere observed (see data analysis). Only one cumulative concentration–response curve for ANG II was obtained in each aortic ring.

2.5. Drugs

The following drugs were used: L-phenylephrine hydrochloride,angiotensin II, acetylcholine chloride, L-NAME, losartan potassium,and PD123319 ditrifluoroacetate (Sigma Chemical Company; St. Louis,MO, USA). Drugs were dissolved in distilled water and subsequentdilutions made in assay buffer.

2.6. Data analysis

Results are expressed as the mean±SEM for the number of aorticrings (n) obtained from four to six different animals. Contractions areexpressed in grams (g) of developed force. Relaxations are expressedas the reduction of the maximum increment in tension (precontrac-tion) obtained by phenylephrine addition. The negative logarithms ofthe molar concentrations of ANG II required to produce 50% of themaximal response (−log EC50) were calculated (by nonlinear re-gression analysis) for each individual concentration–response curvewith a computer program (Graph Pad Software; San Diego, CA, USA).Agonist concentration ratios (CR) were determined from EC50 valuesand, where appropriate, estimates of pA2 obtained from the equation:

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Fig. 1. Concentration–response curves for angiotensin II (ANG II) obtained in ratthoracic aortic ring preparations in the presence (Endo +) and absence (Endo −) ofendothelium, and in the presence of endothelium and NG-nitro-L-arginine methylester (Endo +; L-NAME 100 μM). In most tissues, two (1st and 2nd) concentration–response curves to ANG II were obtained with an interval of 1 h. Results are expressedas grams (g) of developed force. Values are means±SEM (n=8–12). For statisticalanalysis, only the monophasic uphill of concentration–response curves wasconsidered.*Pb0.05 Endo − (1st) vs. Endo + (1st); ФPb0.05 Endo +, L-NAME100 μM vs. Endo + (1st). #Pb0.05 Endo + (2nd) vs. Endo + (1st). #Pb0.05 Endo −(2nd) vs. Endo − (1st).

31T. Pérez et al. / Vascular Pharmacology 54 (2011) 29–35

pA2=log (agonist CR-1)− log [antagonist] (Arunlakshana and Schild,1959). Considering that ANG II elicits biphasic responses in aortictissues, the monophasic uphill (until maximum) of the concentra-tion–response curve to ANG II was used for Schild analysis in thisgroup of experiments.

Statistical analysis was done, as appropriate, by using one-way andtwo-way analysis of variance (ANOVA) followed by Bonferroni orTukey post-hoc tests, in that order (Prism version 4.0; Graph PadSoftware; San Diego, CA, USA). In all comparisons a P value b0.05 wasconsidered as statistically significant.

3. Results

3.1. ANG II induced biphasic responses in aortic rings

In endothelium-intact and endothelium-denuded aortic tissues,ANG II elicited biphasic concentration–response relationships (Fig. 1);that is, ANG II caused concentration-dependent contractions thatgradually reached a maximal level and then diminished amplitude,apparently, with higher concentrations of the agent (Fig. 1). However,in additional experiments designed to evaluate the temporal course ofANG II responses, low (1×10−8 M) as well as high (1×10−5 M)concentrations of this peptide evoked biphasic responses in aorticrings with and without endothelium (Fig. 2). Thus, the biphasiccharacter of ANG II-induced responses can, in principle, be attributedto the loss of tension with time.

Fig. 1 also shows that the concentration–response curves for ANGII were significantly shifted to the left and the maximal responseswere substantially enhanced in endothelium-denuded and L-NAME-treated (100 μM) aortic rings. For this analysis, only the monophasicincrease in tone of the concentration–response curves to ANG II(1×10−10 to 1×10−7 M) was considered.

3.2. Desensitization of ANG II-induced biphasic responses

In endothelium-intact and endothelium-denuded aortic ringpreparations, ANG II-induced biphasic concentration–response curveswere significantly depressed when repeated 1 h after the firstconcentration–response curves had been constructed (Fig. 1). Like-wise, in aortic rings with and without endothelium, the biphasicresponses produced by single concentrations of ANG II wereconsiderably diminished when repeated 1 h after the first responseshad been elicited (Fig. 2). In these experiments, the maximal valuesfor ANG II 1×10−8 M in endothelium-intact and endothelium-denuded aortic tissues, achieved only 65.21±9.49 and 44.96±5.04% of that of the first ones, respectively; whereas for ANG II1×10−5 M the corresponding values were 12.05±3.46 and 8.36±3.77% of that of the first ones, respectively (Fig. 2).

In comparative experiments, two cumulative concentration–response curves to phenylephrine (1×10−9 to 1×10−5 M) wereconstructed in endothelium-intact and endothelium-denuded aorticring preparations. The data showed that the second concentration–response curves to phenylephrine constructed in aortic tissues with(−log EC50, 7.07±0.06, maximum contraction, 1.25±0.04 g, n=12)and without endothelium (−log EC50, 7.30±0.10, maximum con-traction, 1.51±0.06 g, n=11) were not significantly modified whenrepeated 1 h after the first ones (−log EC50, 7.19±0.06, maximumcontraction, 1.32±0.04 g, with endothelium; and −log EC50, 7.31±0.15, maximum contraction, 1.41±0.08 g, without endothelium).

3.3. ANG II provoked additional increases—but not decreases—inphenylephrine-induced active tone

ANG II produced biphasic concentration–response curves onphenylephrine preconstricted endothelium-intact and endothelium-denuded aortic rings (Fig. 3). Specifically, ANG II caused supplemen-

tary increases in tone followed by decreases in tone up to the baselinecontraction (plateau) induced by phenylephrine (Fig. 3). Likewise, asingle high concentration of ANG II (1×10−5 M) evoked biphasicresponses in endothelium-intact and endothelium-denuded aorticrings precontracted with phenylephrine (1×10−7 M), which did notdecrease the active tone induced by phenylephrine (Fig. 5). Controlexperiments showed that the precontraction induced by phenyleph-rine remained stable for at least 1 h (data not shown).

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Fig. 2. Temporal course of a single concentration–response for angiotensin II (ANG II), either 1×10−8 or 1×10−5 M, obtained in rat thoracic aortic ring preparations in the presence(Endo +) and absence (Endo−) of endothelium. Each concentration of ANG II was applied twice (1st and 2nd) to each tissue with an interval of 1 h. Results are expressed as grams(g) of developed force. Values are means±SEM (n=8–10). #Pb0.05 Endo − (2nd) vs. Endo − (1st); *Pb0.05 Endo + (2nd) vs. Endo + (1st).

32 T. Pérez et al. / Vascular Pharmacology 54 (2011) 29–35

3.4. Effect of losartan and PD123319 on the biphasic responses producedby ANG II in aortic tissues pre-contracted with phenylephrine

The ANG II-induced biphasic responses in endothelium-intact andendothelium-denuded aortic rings precontracted with phenyleph-rine, were abolished in the presence of losartan (100 nM) (Fig. 3). It isimportant to note that there were no relaxing responses to ANG II inthe presence of the AT1 blocker (Fig. 3). Also, the effect of PD12319(1 μM) was tested against the response to ANG II in phenylephrine-precontracted endothelium-intact rings. This antagonist did notmodify the concentration–response curves for ANG II (Fig. 4).

3.5. Effects of angiotensin receptor antagonists on the concentration–response relationships of ANG II

In endothelium-intact aortic ring preparations, losartan (10, 30,and 100 nM) concentration dependently shifted the concentration–response curves of ANG II to the right, without reducing the maximalresponse; yielding a pA2 value of 8.19±0.08 and Schild slope of0.98±0.13 (Fig. 6). The slope of the Schild plot was not statisticallydifferent from the theoretical value of unity. On the other hand, theselective AT2 receptor antagonist, PD123319 (0.1, 1, and 10 μM), didnot significantly influence the concentration–response curves forANG II (Fig. 7).

4. Discussion

To our knowledge, this is the first report that analyses functionallythe relative participation of AT2 receptors, and heterologous receptordesensitization induced by AT1 receptors, in the putative relaxationproduced by ANG II in normal rat aorta. The present data show that

ANG II evoked biphasic responses in rat thoracic aorta with andwithout endothelium, characterized by an initial increase followed bya decrease in active tone development. Basically, an agonist that elicitsa biphasic concentration–response relationship may cause contrac-tions to reach a maximal level that subsequently diminishes withhigher concentrations of the agent. An important feature of thebiphasic responses to ANG II, however, is that they were qualitativelythe same (i.e., biphasic) when elicited by a single low or highconcentration of this agonist. Therefore, the ANG II-induced biphasicresponses do not appear to reflect the presence of functionallyseparate high- and low-affinity angiotensin receptors. The ANG II-induced biphasic responses can primarily be associated with the lossof tension with time.

In essence, the desensitization to ANG II could be homologous orheterologous depending on whether the loss of response involvesexclusively angiotensin receptors or, on the other hand, involves thesimultaneous impairment of agonist responsiveness at other, differ-ent, family of receptors (e.g., α1-adrenergic receptors) (Kelly et al.,2008; Kohout and Lefkowitz, 2003;). The biphasic responses inducedby ANG II cannot be explained as a result of heterologous desensiti-zation for the following reason: therewas no relaxing response evokedby cumulative, or single, concentration addition of ANG II onphenylephrine preconstricted endothelium-intact and endothelium-denuded aortic rings; even though, ANG II was administered during aperiod (up to 1 h) several times larger (approximately, 3-fold) thanthe duration of the biphasic responses. In brief, ANG II-inducedreceptor desensitization did not cause a simultaneous loss ofresponsiveness to phenylephrine. By exclusion, our results suggestthat ANG II induced rapid homologous desensitization in aortic tissues.

One aim of this study was to determine the potential contributionof AT2 receptors to ANG II-mediated relaxation in normal rat aorta.

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Fig. 3. Effects of losartan on the concentration–response curves to angiotensin II (ANGII) in endothelium-intact (Endo+) and endothelium-denuded (Endo −) rat thoracicaortic rings preconstricted with phenylephrine (PHE, 1×10−7 M). Aortic rings weretreated with losartan (100 nM) 30 min before the concentration–response curves toANG II were constructed. Results are changes of tone induced by phenylephrine. Datarepresent the means±SEM (n=8). All data are expressed as grams (g) of developedforce. *Pb0.05 vs. Control.

PHE 1x10-7 M

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Fig. 5. Temporal course of a single concentration–response for angiotensin II (ANG II),1×10−5 M, obtained inendothelium-intact (Endo+)andendothelium-denuded (Endo−)rat thoracic aortic rings preconstricted with phenylephrine (PHE, 1×10−7 M). Results arechanges of tone induced by phenylephrine. Values are means±SEM (n=8). All data areexpressed as grams (g) of developed force.

33T. Pérez et al. / Vascular Pharmacology 54 (2011) 29–35

Taken into account previous studies (Arun et al., 2004; Fukada et al.,2005), unexpectedly, we found no evidence of ANG II-induced re-laxation in endothelium-intact and endothelium-denuded aortictissues pre-contracted with phenylephrine. In this series of experi-

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Fig. 4. Effects of PD123319 on the concentration–response curves to angiotensin II(ANG II) in endothelium-intact (Endo+) rat thoracic aortic rings preconstricted withphenylephrine (PHE, 1×10−7 M). Aortic rings were treated with PD123319 (1 μM)30 min before the concentration–response curves to ANG II were constructed. Resultsare changes of tone induced by phenylephrine. Data represent the means±SEM(n=8). All data are expressed as grams (g) of developed force.

ments, ANG II-induced biphasic responses were abolished by theselective AT1 antagonist losartan (100 nM), but relaxation was notobserved. On the other hand, in phenylephrine-precontracted aorticrings with endothelium, PD123319 (1 μM) did not influence theconcentration–response curves for ANG II. Thus, an AT2 receptor-dependent decrease in the contractile response to ANG II was notestablished. At this point, it is important to emphasize that ANG II-induced biphasic responses—in concentrations as high as 1×10−5 M—

did not decrease the active tone induced by phenylephrine in aortictissues. In consequence, the ANG II-induced relaxation and thelosartan-unmasked AT2 receptor-mediated vasodilatation previouslyreported (Arun et al., 2004; Fukada et al., 2005), were not confirmedin the aorta. We do not find an obvious explanation for this dis-crepancy. In consequence, more investigation is needed to elucidatethis problem.

At last, the ANG II-induced concentration–response curves inendothelium-intact rat thoracic aorta were concentration dependent-ly antagonized by losartan, but they were not altered by the AT2antagonist PD123319, indicating that the entire response is mediatedby AT1 receptors. To perform the Schild analysis, we considered onlythe monophasic ascending phase of the concentration–responsecurves to ANG II. These ANG II concentration–response curves wereshifted to the right—without reducing the maximum response—bylosartan. The pA2 value for losartan against the actions of ANG II wascharacteristic of an effect on the AT1 receptors (Smith et al., 1992). Theslope of the Schild plot was not statistically different from thetheoretical value of unity, which is indicative that the receptorpopulation tested was homogeneous (Arunlakshana and Schild,1959). Thus, the data conform to a model of simple competition.Further, PD123319 did not shift ANG II concentration–response

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Fig. 6. (Upper) Losartan antagonism of angiotensin II (ANG II)-induced concentration–response curves in endothelium-intact rat thoracic aortic rings. Data represent means±SEM (n=12), and are expressed as grams (g) of developed force. (Lower) Schild plot.For the Schild analysis, the monophasic uphill (until maximum) of each concentration–response curve to ANG II was used. Given are means±SEM (n=12).

34 T. Pérez et al. / Vascular Pharmacology 54 (2011) 29–35

curves (neither to the right nor to the left) with a concentration rangethat spans two orders of magnitude, and that includes the dissociationconstant (KD) value reported for this antagonist in its interaction withAT2 receptors (Siemens et al., 1994). Hence, under AT2 blockade by

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Fig. 7. Concentration–response curves for angiotensin II (ANG II) obtained inendothelium-intact rat thoracic aortic rings in the absence or presence of differentconcentrations of PD123319. Data represent means±SEM (n=8), and are expressedas grams (g) of developed force.

PD123319, the contractile effect of ANG II was not overexposed innormal rat aorta, as has been described for this vessel under path-ological conditions such as diabetes and hypertension (Arun et al.,2004; Cosentino et al., 2005). Finally, removal of the endothelium(and in some experiments L-NAME) significantly enhanced ANG IIresponses in aortic rings, confirming that NO counteracts thecontraction induced by AT1 receptors (Li et al., 1995).

Recently, it became evident that under some pathologic conditionsthe AT2 receptor level was increased in rat and mice aortas (Arunet al., 2004; Cosentino et al., 2005; Yayama et al., 2006). This evidencesuggests that AT2 receptors contribute to the regulation of vasomotortone by offsetting, via the bradykinin/nitric oxide/cyclic guanosinmonophosphate (BK/NO/cGMP) pathway, the contractile effects ofANG II mediated by the AT1 receptors. On the other hand, otherstudies proposed that the NO system is required to maintain arterialelasticity in conductance vessels (Fitch et al., 2001; Safar and Laurent,2003; Susic et al., 2001). These works have indicated that dilatingarteries with NO increases compliance, whereas the activation ofsmoothmusclewith contractile agonists reduces vascular compliance.Thus, stimulation of up-regulated AT2 receptors by ANG II is predictedto have an important regulatory effect in controlling aorta complianceunder conditions associated with vascular tissue damage. However,we found that the AT2 receptors do not contribute to the regulation oftone by counteracting the contractile effects of ANG II in normal rataorta. In fact, the depression of ANG II-induced contractile responsesin rat aorta (with and without endothelium) occurred as aconsequence of AT1 receptors desensitization. This desensitization isan adaptive response used by cells to prevent the detrimental effectsthat can result from persistent receptor stimulation Kohout andLefkowitz, 2003). Thus, desensitization of the potent contractile effectof ANG II (and other endogenous agonists) may be a protectivemechanism to avoid important decreases in vascular (aortic) com-pliance. Increased arterial stiffness may lead to impaired perfusion ofthe peripheral organs and augmented cardiac afterload (Kameyamaet al., 2005).

5. Conclusion

The results of the present investigation indicate that ANG II elicitedbiphasic responses mediated by AT1 receptors in aortic rings, whichcan be attributed to AT1 receptors activation and desensitization withtime. Rapid desensitization induced by ANG II proved to behomologous in nature, since precontractile tone induced by the α1-adrenergic agonist phenylephrine, was not depressed by ANG II (i.e.,ANG II did not induce heterologous α1-adrenergic receptor desensi-tization). We found no functional evidence of the participation of AT2receptors in ANG II-induced biphasic effects in normal rat aorta. ANGII does not exert relaxant effects in normal rat aorta.

Acknowledgements

This workwas supported by Secretaría de Investigación y Posgradodel Instituto Politécnico Nacional (SIP-IPN); Comisión de Operacióny Fomento de Actividades Académicas del IPN (COFAA); and theConsejo Nacional de Ciencia y Tecnología (CONACYT). The authorsthank Ms. Lourdes Ramírez Pichardo for technical assistance.

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