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Cardiovascular Research 2005 65(2):478-486; doi:10.1016/j.cardiores.2004.10.007
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Copyright © 2004, European Society of Cardiology

Modulatory role of the adventitia on noradrenaline and angiotensin II responses

Role of endothelium and AT2 receptors

Beatriz Somozaa, M. Carmen Gonzáleza, José María Gonzáleza, Fatima Abderrahim, Silvia M. Arribasa and María S. Fernández-Alfonsob,*

aDepartamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain
bDepartamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid, Spain

* Corresponding author. Unidad de Cartografía Cerebral, Instituto Pluridisciplinar, Paseo Juan XXIII, no. 1, Madrid 28040, Spain. Tel.: +34 91 394 3261; fax: +34 91 394 3265. Email address: marisolf{at}farm.ucm.es

Received 29 July 2004; revised 6 October 2004; accepted 7 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: We have studied the modulatory role of the adventitia on vascular tone and nitric oxide (NO) availability in response to noradrenaline (NA) and angiotensin II (Ang II).

Methods: Changes in isometric tension were determined in carotid arteries from 3-month-old Sprague–Dawley rats denuded from adventitia (–A) and compared to intact rings (+A). NO availability was assessed by the fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2).

Results: Responses to NA (10–10 to 10–6 M) were: (i) significantly lower in –A compared to +A rings; (ii) equally enhanced in +A and –A rings without endothelium; and (iii) reduced in +A and –A rings incubated with superoxide dismutase (SOD; 15 U/ml). Responses to Ang II (10–10 to 10–7 M) were: (i) similar between +A and –A segments; (ii) equally reduced in both groups by SOD; and (iii) increased by endothelial denudation in both +A and –A arteries. Blockade of AT2 receptors with PD 123,319 (10–7 M) significantly increased Ang II-induced contractions in +A rings. In segments preincubated with losartan (10–5 M) and precontracted with NA (10–7 M), Ang II elicited a relaxation that was abolished by L-NAME (10–4 M), PD 123,319 (10–7 M), and endothelium or adventitial removal. NO availability was increased in carotid rings stimulated with Ang II, but not with NA. This NO release was blocked by PD 123,319 (10–7 M) and endothelium denudation.

Conclusions: These results suggest that the adventitia differently modulates responses to vasoconstrictors and that it is a key layer in Ang II-induced contractions, mediating NO release from the endothelium via AT2 receptors. This increase in NO counterbalances basal superoxide release.

KEYWORDS Adventitia; Angiotensin II; AT2 receptors; Noradrenaline; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
From the three vascular layers that form the arterial wall (adventitia, media, and intima), the adventitia has been considered until recently only as a structural support for the media underneath, receiving for itself minor attention as a potential modulator of vascular tone. Most studies on the adventitia have focussed on remodeling of this layer in some pathological conditions, such as neointima formation [1–5], hypertension [6,7], and atherosclerosis [7,8]. However, there is increasing evidence of a direct participation of the adventitia on blood vessel function, as recently reported by our group and others [9–11]. One of the reasons that less attention has been paid on adventitial modulation of vascular tone might be the difficulty of obtaining separate layers for performing functional studies. Recently, Schulze-Bauer et al. [10] have developed a method for obtaining separate rings of adventitia from human femoral arteries. This technique is, however, not feasible in arteries of experimental animal models [10]. In this regard, our group has developed a method for removing the adventitia, which allows the study of its role on rat artery function [11].

The adventitia is a source of superoxide anions [12] due to the presence of an NADP(H) oxidase [13]. In fact, the primary site of superoxide production in the vessel wall from rabbit [13], rat [14,15], and human arteries [16] is the adventitia. This constitutes a significant scavenger for nitric oxide (NO) reaching the smooth muscle [14]. The interaction between NO and superoxide, both reactive oxygen species, occurs rapidly [17] and induces a loss in NO availability and action, such as endothelium-dependent relaxations [18].

Angiotensin II (Ang II) plays a major role in hypertension and other cardiovascular diseases. This peptide induces a direct contractile effect on smooth muscles mediated by the AT1 receptor [19]. Increasing evidence suggests that the contractile effect is opposed by AT2 receptor-mediated increase in NO availability [20–25]. The fact that AT2 receptors predominate in the adventitia of certain arteries [26,27] leads to the hypothesis of a modulatory role of this layer on Ang II-induced contractions through a regulation of the balance between NO and superoxide anions. Since other vasoconstrictors, like noradrenaline (NA) do not seem to stimulate NO release from the adventitia, we have compared the role of this layer on contractile response, as well as NO availability, in response to Ang II and NA.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
Three-month-old rats (Harlan Sprague–Dawley under specific pathogen-free conditions; weight 350–400 g) were used. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Rats were anesthesized with sodium pentobarbital (50 mg/kg) and bled by cardiac puncture. Carotid arteries were carefully isolated, placed in oxygenated physiological salt solution (PSS), and cleaned of blood and perivascular fat. PSS had the following composition: 115 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.01 mM EDTA, 11 mM glucose, and 5 µM indomethacin to avoid prostaglandin-mediated effects. Since NO availability depends on the balance between NO and superoxide production, all experiments were performed in the presence of 0.8 µM dexamethasone to avoid iNOS induction.

2.1. Functional studies
Functional studies were performed in carotid artery rings with (+A) and without (–A) adventitia. Adventitia removal was performed as previously described [11]. Briefly, arteries were placed in a shaking bath at 37 °C for 15 min in PSS containing 2 mg/ml collagenase type II (chlostridiopeptidase A; EC 3.4.24.3 [EC] ). Thereafter, vessels were immediately rinsed and placed for 10 min in PSS at 4 °C. The arteries were then fixed with pins at both ends to a Sylgard based dissecting dish containing cold PSS, and the adventitia was carefully removed by gentle peeling with two pairs of fine forceps under a dissecting microscope.

In order to determine vascular function, 3-mm-long carotid artery rings were suspended on two intraluminal parallel wires, introduced in an organ bath containing PSS and connected to a Piodem strain gauge for isometric tension recording. Segments were given an optimal resting tension of 1.5 x g, which was readjusted every 15 min during a 90-min equilibration period. Thereafter, the vessels were exposed to 75 mM KCl to check their contractility, and concentration–response curves to NA (10–10 to 10–6 M) or Ang II (10–10 to 10–7 M) were determined. Endothelial integrity was analyzed by addition of acetylcholine (ACh) to segments precontracted with 10–7 M NA. In some arteries (both +A and –A rings), the endothelium was removed by gentle scraping with a cotton thread through the vessel lumen. Segments with more than 60% relaxation to 10–5 M ACh were considered with endothelium (+E) and segments with less than 10% relaxation to 10–5 M ACh were considered as endothelium-free (–E). In another set of experiments, segments were precontracted with 10–7 M NA in the presence of losartan (10–5 M), and Ang II (10–8 and 10–7 M) was added to both +A and –A rings. At least three arterial segments from each animal were used per group.

2.2. Determination of NO availability by confocal microscopy
NO availability was determined by the fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2) [28]. This compound enters the cells where it is hydrolysed by cytosolic estearases and trapped in the cytosol. The fluorescent chemical transformation of DAF-2 is based on the reactivity of aromatic vicinal diamines with NO in the presence of dioxygen. This N-nitrosilation of DAF-2 produces a highly green fluorescent triazole form, DAF-2T, which can be visualized with the 488/515-nm line of the microscope. Because this nitrosilation is essentially irreversible [29], DAF-2 fluorescence reflects a sum total of NO availability and has been used to monitor in situ the NO production in arteries [30]. Intact carotid arteries were cut in rings of 50 µm. Thereafter, rings were stabilized in PSS containing 0.8 µM dexamethasone for 30 min at 37 °C and stained with DAF-2 (10–5 M) dissolved in PSS for 30 min. DAF-2 was incubated in the darkness at room temperature in a shaking bath. In order to avoid NO inactivation by superoxide anion, all the rings (controls and rings incubated with either NA, Ang II, PD 123,319, or both) were incubated with SOD (15 U/ml) throughout the experimental procedure. DAF-2-negative controls were also incubated in 10–4 M L-NAME (N{omega}-nitro-L-arginine methyl ester) throughout the experimental period. Following the 30-min DAF-2 incubation period, rings were washed in PSS and mounted on slides for visualization with a Leica TCS SP2 confocal system (Leica Microsystems, Germany) fitted with argon and helium–neon laser sources and coupled to a Leica DMIRE 2 microscope, using the 488/515-nm line of the microscope. Stacks of 15 serial optical sections (1 µm thick) were captured from each ring with a x 40 oil objective (NA 1.25), x 2 zoom, and a maximal projection was then obtained. All images from control and stimulated rings were captured under identical conditions of laser intensity, brightness, and contrast. Adventitial cells with an intensity level larger than 100 brightness per pixel were considered stained with DAF-2. These cells were counted in several areas of the ring and averaged (number of cells per unit area).

2.3. Analysis of data
Contractions are expressed as the percentage of contraction produced by 75 mM KCl. Relaxations are expressed as the percentage of contraction produced by NA 10–7 M. The maximum response (Emax values) and the negative logarithm of concentrations of NA or Ang II producing 50% of maximum response (pD2 values) were calculated by a nonlinear regression analysis of each individual concentration–response curve using GraphPad Prism Software (San Diego, CA, USA). Statistical significance was analyzed by ANOVA. p<0.05 was considered significant.

2.4. Reagents
Drugs and reagents were obtained from Sigma (St. Louis, MO, USA). Drugs were dissolved in distilled water. Indomethacin was dissolved in 5% NaHCO3.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Effect of adventitia on NA-induced contractions
In adventitial-denuded vessels, NA (10–10 to 10–6 M) induced significantly lower contractions in efficacy than in control rings (Fig. 1A; Table 1). To determine if this effect was due to adventitial-derived superoxide anions, concentration–response curves to NA were performed in the presence of superoxide dismutase (SOD; 15 U/ml), which induced a significant shift to the right of concentration–response curves to NA in both +A and –A segments (Fig. 1B; Table 1). However, the shift in pD2 elicited by SOD (Table 1) was more pronounced in segments with adventitia than in segments without adventitia. This result suggests that the adventitia is the major source of superoxide anions in the vascular wall, but that there is also a small but significant contribution from the other layers.


Figure 1
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  		Fig. 1 (A) Effect of adventitial denudation (–A) on concentration–response curve to NA in carotid arteries. (B) Effect of SOD (15 U/ml) on the concentration–response curve to NA in carotid arteries with adventitia (+A; right panel) and without adventitia (–A; left panel). (C) Effect of endothelium denudation on concentration–response curves to NA in carotid arteries with adventitia (+A; right panel) and without adventitia (–A; left panel). Contractions are expressed as percentage of a previous contraction to 75 mM KCl. Results are mean ± S.E.M. The number of animals is indicated in parentheses. *p<0.05; ***p<0.001 within graph. A detailed statistical analysis is shown in Table 1.

 

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  		Table 1 Role of the adventitia on NA-induced contractions

 
The possible interrelationship between the endothelium and the adventitia was analyzed by using +E and –E arteries. The difference in NA contractions between +A and –A arteries was abolished by endothelium removal (Fig. 1C; Table 1), suggesting that adventitial superoxide release was affecting endothelial NO availability.

3.2. Effect of adventitia on Ang II-induced contractions
We next examined the role of adventitia on the response elicited by Ang II (10–10 to 10–7 M) in rat carotid arteries. In endothelium-intact rings, Ang II induced contractions from 3 x 10–10 M and reached the maximal response at 10–7 M. Higher concentrations elicited desensitization of the blood vessels, both in +A and –A segments (data not shown). In contrast to the results described for NA, no differences were observed in concentration–response curves to Ang II between adventitia-denuded segments and control rings (Fig. 2A; Table 2). This result suggests that Ang II stimulates, via the adventitia, the release of some factor that counterbalances the previously described basal superoxide production.


Figure 2
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  		Fig. 2 (A) Effect of adventitial denudation (–A) on concentration–response curve to Ang II in carotid arteries. (B) Effect of SOD (15 U/ml) on the concentration–response curve to Ang II in carotid arteries with adventitia (+A; left panel) and without adventitia (–A; right panel). (C) Effect of endothelium denudation on concentration–response curves to Ang II in carotid arteries with adventitia (+A; left panel) and without adventitia (–A; right panel). (D) Effect of PD 123,319 (10–7 M), a selective AT2 receptor antagonist, on the concentration–response curve to Ang II on carotid arteries with adventitia (+A; left panel) and without adventitia (–A; right panel). Contractions are expressed as percentage of a previous contraction to 75 mM KCl. Results are mean ± S.E.M. The number of animals is indicated in parentheses. *p<0.05; ***p<0.001 within graph. A detailed statistical analysis is shown in Table 2.

 

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  		Table 2 Role of the adventitia on Ang II-induced contractions

 
In the presence of SOD (15 U/ml), there was a similar decrease in the efficacy of Ang II in both +A and –A segments (Fig. 2B; Table 2). In addition, endothelium denudation induced an increase in efficacy and sensitivity in both +A and –A arteries compared to control rings (Fig. 2C; Table 2), but no differences between segments with and without adventitia were observed (Table 2), suggesting that the counterregulatory effect of the adventitia on Ang II-induced contractions was dependent on NO release from the endothelium.

Since the AT2 receptor has been suggested to induce an increase in NO availability [20–25], the participation of this receptor was assessed with PD 123,319, a selective AT2 receptor antagonist. PD 123,319 (10–7 M) did not modify baseline tension, but it significantly increased the Emax to Ang II in the presence of adventitia (Fig. 2D; Table 2), suggesting a role for adventitial AT2 receptors on the modulation of Ang II responses through NO release. In –A segments, PD 123,319 (10–7 M) induced a slight but significant shift to the left of the concentration–response curve to Ang II (Fig. 2D; Table 2), indicating that endothelial AT2 receptors also participate in NO release.

3.3. Effect of adventitia on Ang II-induced relaxations
In order to confirm AT2-mediated NO release, responses to Ang II were analyzed in segments precontracted with NA (10–7 M) and preincubated with losartan (10–5 M) to block AT1 receptors. Under these conditions, Ang II (10–8 and 10–7 M) elicited a concentration-dependent vasodilatory effect (Table 3). This dilatation was markedly reduced by PD 123,319 (10–7 M) and abolished in the presence of L-NAME (10–4 M) and adventitial or endothelium removal (Table 3).


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  		Table 3 Relaxation induced by Ang II in segments preincubated with losartan (10–5 M) to block AT1 receptors and precontracted with NA (10–7 M) in carotid arteries with (+A) and without (–A) adventitia and in the presence of different agents

 
3.4. Role of adventitia NO production
To support the functional study, we investigated next whether the AT2 receptor was stimulating NO production in rat carotid artery segments, using the fluorescent NO indicator, DAF-2. In order to exclude the interaction with superoxide anions, all segments were incubated simultaneously with SOD (15 U/ml). Control segments showed DAF-2 fluorescence throughout the vascular wall, being the intensity highest in the endothelium and in some adventitial cells. In the presence of L-NAME, green fluorescence was negligible in the artery ring with the exception of the elastic lamellae, which have autofluorescent properties at the wavelength used (data not shown). NA-stimulated rings (10–6 M) showed similar fluorescence levels as control rings. Ang II (10–7 M) induced a robust increase in NO production, when compared to control. Increase in fluorescence was detected mainly in the adventitia. DAF-2 increase in fluorescence induced by Ang II was significantly reduced by PD 123,319 (10–7 M), which had no effect per se (result not shown) and was abolished by endothelial denudation (Fig. 3A). Quantification of the number of stained adventitial cells showed a 10-fold increase after Ang II stimulation, but not after NA stimulation. The number of stained cells was reduced after preincubation with PD 123,319 (10–7 M) and abolished by endothelial denudation (Fig. 3B).


Figure 3
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  		Fig. 3 (A) Confocal projections showing in situ NO generation in carotid artery rings stained with the fluorescent NO indicator, DAF-2 diacetate, and the effect of stimulation with NA (10–6 M), Ang II (10–7 M), or Ang II (10–7 M) plus PD 123,319 (10–7 M) and endothelial denudation. All rings were incubated throughout the experiment with 15 U/ml SOD to avoid interaction with superoxide anions. Images were captured under identical conditions of brightness, contrast, and laser intensity. E=endothelium; M=media; A=adventitia. (B) Quantification of the number of adventitial cells stained with DAF-2 (n=4 rats). *p<0.05; **p<0.001 when compared to control; #p<0.001 when compared to Ang II alone.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The present study confirms our previous work suggesting that the adventitia exerts a modulatory role on vascular function [11]. In addition, we demonstrate that this layer plays a key role in the responses to Ang II by counterbalancing basal superoxide production with the enhancement of NO release through an AT2-dependent mechanism.

Basal superoxide production in the vessel wall has been detected in the adventitia of rabbit [13], rat [14,15], and human arteries [16]. Previous data suggested that this superoxide release from the adventitia might reduce endothelial NO availability [11,12]. In accordance, the lower contraction elicited by NA in adventitial-denuded vessels and the abolishment of this difference by endothelium removal, shown in the present study, suggest an involvement of adventitial superoxide anions in the reduction of NO availability. This was confirmed by the rightward shift of the concentration–response curve to NA elicited by SOD. The fact that the concentration–response curve to NA plus SOD is shifted to the right in both +A and –A carotid arteries indicates that, although the adventitia seems to be the major source for basal superoxide production, the contribution of other layers cannot be excluded.

Interestingly, the response to Ang II is affected in a different manner by the adventitia. In contrast to NA-induced responses, adventitial removal did not modify concentration–response curves elicited by Ang II. This result was apparently suggesting that either adventitial superoxide anion was not playing a role on Ang II-induced contractions, or that Ang II was stimulating via the adventitia the release of some factor that counterbalanced the previously described basal superoxide production. The latter possibility was endorsed by the results obtained in the presence of SOD or in absence of endothelium. The significant reduction of Ang II contractions by SOD in segments without adventitia confirmed that other vascular layers of the rat carotid artery were releasing superoxide anions. In addition, our results with the NO indicator, DAF-2 diacetate, also indicated that Ang II stimulated NO release.

It is important to note that, in our experimental conditions, Ang II is not stimulating superoxide production in the rat carotid artery, as has been demonstrated by several authors [33–37]. Firstly, the concentration–response curve to Ang II is performed in approximately 30 min, which is not time enough for NADP(H) oxidase induction, which requires at least 3 h [36]. Secondly, all experiments have been performed in the presence of dexamethasone in order to avoid the inducible form of the NO synthase. Under these conditions, NADP(H) oxidase induction has been demonstrated to be also inhibited [38].

Since several authors have suggested that Ang II indirectly increases NO production by stimulating AT2 receptors [20–25], an attractive hypothesis is that Ang II stimulates the production of NO via AT2 receptors of the adventitia. The present results give direct evidence for endothelial NO release through AT2 receptors located in the adventitia. This was confirmed, firstly, by the functional data showing that in the presence of adventitia, PD 123,319 induced an increase in Emax in response to Ang II. A second evidence for the stimulation of NO release through AT2 receptors was obtained by the relaxation experiments in carotid artery rings incubated with losartan. The direct confirmation was obtained with DAF-2 experiments, which showed that Ang II-increased NO availability was abolished by AT2 receptor blockade. Interestingly, although the present results demonstrate that mainly AT2 receptors from the adventitia are responsible for NO release and, therefore, for the modulation of vascular tone in response to Ang II, AT2 receptors from the endothelium also seem to have a significant role. The precise mechanism of AT2-mediated NO increase is still unclear. It has been proposed that AT2 stimulation involves the release of bradykinin, which would stimulate endothelial NO release through the activation of bradykinin B2 receptors [20–23,25].

One intriguing question is the source of NO after AT2 receptor stimulation. Our results demonstrate that the most likely source is the endothelium. The endothelium produces a combination of vasodilatory factors in rat carotid arteries (i.e., NO and prostacyclin) [32]. We excluded the participation of the latter by the use of indomethacin in the organ bath during the whole experiment. On the other hand, DAF-2 experiments showed a complete abolishment of Ang II-stimulated fluorescence in the absence of endothelium. This result confirmed the essential role of this layer on AT2 receptor-stimulated NO release and excluded a possible contribution of endothelial cells from the vasa vasorum in the adventitia. However, when analysing fluorescence in response to Ang II, a 10-fold increase was detected in adventitial cells, but not in the medial layer. We do not have, at the moment, an explanation for this finding, which suggests a cross-talk between the endothelium and the adventitia that has to be explored in future studies.

The mutual regulation between Ang II and NO is well known [39]. There is increasing evidence for role of the AT2 receptor as a counterregulator of AT1 receptor-mediated vasoconstriction through endogenous NO production [20–25,31,40,41]. This work demonstrates, furthermore, the importance of the adventitia in this balance (Fig. 4). The here reported counterbalance of basal superoxide production by adventitial AT2-mediated NO release might be of special importance when considering the antihypertensive effect of AT1 receptor antagonists. Long-term administration of AT1 blockers increases Ang II plasma levels and allows a stimulation of the unopposed AT2 receptor [42,43]. This is of special relevance, since AT2-mediated relaxation is highly reproducible and does not show desensitization [31], as AT1 receptor-mediated contraction does. In view of these data and together with the present findings, we suggest that AT1 receptor antagonists might have an effect at different levels: (i) direct inhibition of contraction of smooth muscles; (ii) direct blockade of superoxide production in the vessel wall; (iii) increase of NO release through an enhanced stimulation of adventitial AT2 receptors; and (iv) indirect reduction of superoxide production through the increase in NO. Since the distribution of AT2 receptors strongly depends on vascular territory [34,40,41], the proposed superoxide/NO balance might vary from one vascular bed to another.


Figure 4
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  		Fig. 4 Schematic diagram showing the proposed mechanism of adventitial modulation of responses to Ang II. Basal superoxide production in the adventitia reduces endothelial NO availability (right arrow). Ang II stimulates NO release from the endothelium through an AT2-dependent mechanism, mainly in the adventitia but also in the endothelium. The increased NO counterbalances basal superoxide production in the vessel wall (left arrow). E=endothelium; M=media; A=adventitia.

 


   Acknowledgements
 
This work has been supported by FIS 98/0736 and I+D BFI2001-0638 Spain. We are grateful to Carmen Fernández Criado from the animal facility and Diego Megías for his technical assistance with the confocal microscope.


   Notes
 
Time for primary review 40 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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