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Cardiovascular Research Advance Access first published online on August 30, 2007
This version [Corrected Proof] published online on October 4, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm008
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ß-adrenergic relaxation in pulmonary arteries: preservation of the endothelial nitric oxide-dependent ß2 component in pulmonary hypertension

Véronique Leblais1,2,*, Estelle Delannoy1,2, Fleur Fresquet1,2, Hugues Bégueret3, Nadège Bellance1,2, Sébastien Banquet1,2, Cécile Allières4, Lionel Leroux4, Claude Desgranges4, Alain Gadeau4 and Bernard Muller1,2

1 Université Bordeaux 2, Laboratoire de Pharmacologie de l'UFR Pharmacie, Bordeaux F-33076, France
2 INSERM, U885, Bordeaux F-33076, France
3 Hôpital du Haut-Lévêque, Service d'Anatomie et Cytologie Pathologiques, Pessac F-33600, France
4 INSERM, U828, Pessac F-33600, France

* Corresponding author. Tel: +33 5 57 57 12 01; fax: number: +33 5 57 57 12 01. E-mail address: veronique.leblais{at}phcodyn.u-bordeaux2.fr

Time for primary review: 17 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: ß-adrenoceptor (ß-AR)-mediated relaxation was characterized in pulmonary arteries from normoxic and hypoxic (as model of pulmonary hypertension) mice. The endothelial NO synthase (eNOS) pathway was especially investigated because of its potential vasculoprotective effects.

Methods: Pulmonary arteries from control or hypoxic (0.5 atm for 21 days) wild-type or eNOS–/– mice were used for pharmacological characterization of ß-AR-mediated relaxation in myograph, and for immunohistochemistry using anti-ß-AR antibodies.

Results: In pulmonary arteries from normoxic mice, isoproterenol (ß-AR agonist) and procaterol (selective ß2-AR agonist) elicited relaxation, while cyanopindolol and CL316243 [GenBank] (ß3-AR agonists) were ineffective. The effect of isoproterenol was antagonized by CGP20712A and ICI118551 (ß1- or ß2-AR antagonists, respectively) and also partially inhibited by N{omega}-nitro-L-arginine methylester (L-NAME, a NOS inhibitor), endothelium denudation, or eNOS gene deletion. Relaxation to procaterol was abolished by L-NAME or endothelium removal. In eNOS–/– mice, procaterol-induced relaxation was decreased but was insensitive to L-NAME, this residual effect involving other endothelium-dependent relaxant factors as compensatory mechanisms. Immunostaining for ß2-AR was observed in the endothelial layer, but not the medial layer of pulmonary arteries. Pulmonary arteries from hypoxic mice exhibited decreased endothelial NO-dependent relaxation to acetylcholine. However, in these arteries, relaxation to procaterol was either unaffected (extralobar segments) or even increased (intralobar segments) and was still abolished by L-NAME or endothelium removal.

Conclusion: ß1- and ß2-AR, but not ß3-AR, mediate relaxation of mice pulmonary arteries. The ß2-AR component is dependent on eNOS activity and is preserved following chronic hypoxia. These data highlight the role of the ß2-AR as a pharmacological target to induce/restore endothelial NO-dependent protective effects in pulmonary circulation.

KEYWORDS Adrenergic; Endothelial function; Hypoxia; Nitric oxide; Pulmonary

Received July 2, 2007; revised July 23, 2007; accepted August 5, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Elevation of pulmonary arterial pressure is a common complication and a predictor of mortality in patients with severe chronic obstructive pulmonary disease.1 Remodelling of pulmonary arteries and persistent vasoconstriction contribute to the increase in pulmonary arterial resistance.2 Endothelial dysfunction of pulmonary arteries, with impaired endothelial nitric oxide (NO) production and/or availability, plays a key role in the pathogenesis of this disease.1,3 Since endothelium-derived NO possesses multiple vasculoprotective effects, including inhibition of smooth muscle cell proliferation and contraction,4,5 stimulation of endothelial NO production has potential benefits in hypoxic pulmonary hypertension.

All ß-adrenoceptors (ß-AR), namely ß1- (including its low affinity state), ß2-, and ß3-AR, mediate vasorelaxation, with underlying mechanisms that could be different from one vessel to the other, depending on ß-AR subtypes and their location on endothelial and/or smooth muscle cells. Vasorelaxation is commonly attributed to ß2-AR, which represents the predominant ß-AR subtype in most vascular smooth muscles, and subsequent activation of the cAMP/cAMP-dependent protein kinase (PKA) pathway.6 In systemic circulation, depending on vascular beds, ß-AR-mediated relaxations were described as being completely or partly endothelium-dependent,7–9 or totally endothelium-independent.10,11 The endothelial NO pathway was found to be involved in relaxation elicited by activation of each of the ß-AR subtypes, ß1-,8 ß2-,7,9 and ß3-AR.12

In pulmonary arteries, both ß1- and ß2-AR are expressed.13 The ß-AR-mediated relaxation is partly NO-dependent in extralobar and distal intralobar rat pulmonary arteries,14,15 but not in proximal intralobar segments.16 However, ß-AR subtypes mediating NO-dependent relaxation have not been definitively characterized.14,15 Investigation of the relaxant effect of the non-selective ß-AR agonist, isoproterenol, in pulmonary arteries from transgenic mice lacking ß1- and/or ß2-AR shows that it is exclusively dependent on ß1-AR, but whether endothelial NO contributes to this relaxation was not specifically assessed.17 Pulmonary arteries from hypoxic models usually exhibit decreased endothelial NO-dependent relaxation to acetylcholine.18 The NO-dependent ß-AR-mediated relaxation, although not directly addressed, appears to be only partly reduced in pulmonary arteries from hypoxic rats.15 Thus, further investigations appear necessary to evaluate the role of ß-AR as pharmacological target to induce/restore endothelial NO-dependent effects in pulmonary circulation.

Therefore, the aims and rationale of the present study were as follows: (i) to characterize the ß-AR subtypes mediating relaxation of mice pulmonary arteries; (ii) to determine the contribution of endothelial NO-dependent and -independent mechanisms, and of the cAMP/PKA pathway, to this ß-AR-mediated relaxation; (iii) to investigate the effects of chronic hypoxia on this ß-AR-mediated relaxation, and especially on the endothelial NO-dependent component; (iv) to compare the former features in extra- and intra-lobar segments of pulmonary arteries. Work was conducted using pharmacological approach for characterization of ß-AR-mediated relaxation in endothelium-intact or -denuded pulmonary arteries, genetic approach with mice deleted for eNOS gene (eNOS–/– mice), and immunohistochemistry for ß-AR localization.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
The investigation conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996), with agreement obtained by French authorities (A 33409). Unless otherwise indicated, all drugs and reagents were purchased from Sigma. Experiments were performed in male C57BL6 mice (11–14 weeks old; Elevage Janvier) and male eNOS–/– mice (10–12 weeks old; obtained from Dr Gödecke).19 Some C57BL6 mice were placed for 21 days under hypoxic environment (380 mmHg).18

2.1 Right ventricular hypertrophy and pressure
The weight ratio (right ventricle/left ventricle+septum) was determined for the assessment of right ventricular hypertrophy.18 Right ventricular pressure was also determined in anesthetized (ketamine/xylazine, i.p.), tracheotomized, and artificially ventilated mice. After chest opening, a 24-GA catheter was introduced in the right ventricle, and the heparinized probe of a pressure transducer (Millar instrument Inc.) was inserted in the catheter and pushed inside the ventricle. After 1 min stabilization, pressure values were recorded. Right ventricle weight, left ventricle+septum weight, and mean right ventricular pressure in eNOS–/– mice were 24.2 ± 1.1 mg (n = 11), 89.9 ± 2.4 mg (n = 11), and 14.9 ± 1.6 mmHg (n = 5), respectively. These values were not significantly different from those obtained in wild-type mice, which were 27.6 ± 0.8 mg (n = 60), 87.0 ± 1.5 mg (n = 60), and 16.2. ± 2.2 mmHg (n = 5).

2.2 Vascular reactivity studies
For each mouse, two segments of extralobar (left and right branches) and of intralobar (first-order branch) pulmonary arteries were dissected and mounted in a myograph.18 In some experiments, endothelium was removed using 3-[(3-cholamidopropyl)- dimethylammonio] 1-propanesulfonate (CHAPS).16 Preparations developing a wall tension below 0.5 mN/mm following 80 mM KCl addition were discarded. All relaxant agents studied were applied to arteries which were submaximally pre-contracted with prostaglandin F2{alpha} (PGF2{alpha}, dinoprost tromethamine from Centravet). To avoid possible influence of {alpha}-AR,20 experiments with ß-AR agonists were performed in the presence of the {alpha}-AR antagonist phentolamine (10 µM). Pharmacological agents used for characterization of the ß-AR-mediated relaxation were added 30 min before PGF2{alpha}, their concentration being selected from laboratory or literature data. They did not modify basal tone, except the NOS inhibitor N{omega}-nitro-L-arginine methylester (L-NAME, 300 µM) which increased the tone by 0.04 ± 0.01 mN/mm.

To assess endothelium functionality, relaxation to acetylcholine, which is fully endothelial NO-dependent in mice pulmonary arteries,18 was determined. Relaxation induced by 10 µM acetylcholine was 62.4 ± 1.1% (n = 69) and 1.1 ± 0.4% (n = 30; P < 0.001) in control and CHAPS-treated extralobar arteries, respectively.

To characterize the ß-AR subtypes mediating relaxation, ß-AR agonists were added either in a cumulative manner (isoproterenol, CGP12177, cyanopindolol from Tocris, CL316243 [GenBank] , or BRL37344) or at a single concentration (0.1 µM, producing maximal effect) for procaterol (from Tocris), because cumulative addition of this drug resulted in an unreliable concentration-dependent relaxation. Effects of ß-AR agonists were also studied in the presence of ß-AR antagonists (ICI118551, CGP20712A, and SR59230A).

To determine the contribution of endothelial NO to ß-AR-mediated relaxation, effects of ß-AR agonists were determined in the presence of the NOS inhibitor L-NAME or the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,3a]quinoxalin-1-one (ODQ, 1 µM; from Tocris). In a subset of experiments, to evaluate a possible permissive role of basal eNOS-derived NO,21 arteries were pre-contracted with PGF2{alpha} in the presence of L-NAME, and once contraction was stable, the NO donor [2-(N,N-diethylamino)- diazenolate-2-oxide, DEA-NO; from Alexis] was added at a subthreshold concentration (30 nM, producing about 15% relaxation), followed by procaterol (0.1 µM). In another set of experiments, procaterol was studied in pulmonary arteries from eNOS–/– mice, in the absence or presence of L-NAME or L-NAME combined with inhibitors of other endothelium-derived relaxing factors than NO (10 µM indomethacin for inhibition of cyclooxygenase-derived factors, and 0.1 µM charybdotoxin + 0.1 µM apamin for inhibition of endothelium-derived hyperpolarizing factor, EDHF, both K+ channels blockers being purchased from Latoxan). Relaxation to acetylcholine (as reference endothelial NO-dependent agent) was similarly characterized in arteries from eNOS–/– mice. When responses of arteries from hypoxic mice were compared to those from controls, acetylcholine and sodium nitroprusside (SNP) were studied, as standard agents for assessment of endothelial dysfunction.18

To determine the potential role of the cAMP/PKA pathway in ß-AR-mediated relaxation, effect of ß-AR agonists was studied in the absence or presence of the inhibitor of adenylate cyclase, SQ22536 (300 µM; from VWR) or of PKA, Rp-8-bromoadenosine 3'5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS, 100µM; from Biolog).

2.3 Immunohistochemistry
Sections (4 µM thick) from formalin-fixed, paraffin-embedded pulmonary arteries were processed for immunohistochemistry. Antigen retrieval was carried out in heat citrate buffer pH 6.0 (Dako REALTM Target Retrieval Solution) for 10 min in a streamer. Immunohistochemical study was performed using the biotin- streptavidin-peroxydase LSAB kit (Dako) with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology) directed against ß1-AR (clone V-19, at 1/50) or ß2-AR (clone M-20, at 1/50). Diaminobenzidine complex was used as the chromogen. Negative control slides were treated similarly, by using normal rabbit serum. The sections were counterstained with Mayer's hematoxylin.

2.4 Data analysis
Relaxant effects are expressed as percentage of relaxation of the tone induced by PGF2{alpha}. Data are given as mean±SEM of n experiments (n: number of mice). Concentration-response curves were compared using analysis of variance (ANOVA) for repeated measures. Other parameters were compared using Student's t-test. Differences were considered statistically significant when P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Characterization of the ß-AR subtypes mediating relaxation of mice extralobar pulmonary arteries
In extralobar pulmonary arteries, the non-selective ß-AR agonist isoproterenol induced a relaxation, which was antagonized by ICI118551 (0.1 µM; a selective ß2-AR antagonist; Figure 1A). CGP20712A (0.1 µM; a selective ß1-AR antagonist) further antagonized the effect of isoproterenol (Figure 1A). However, increasing the concentration of CGP20712A to 3 µM [to antagonize the low affinity state (l.a.s.) of ß1-AR] had no additional inhibitory effect (Figure 1A). In the presence of both ICI118551 and CGP20712A, the response to isoproterenol was not further modified by SR59230A (1 µM; a ß3-AR antagonist) (Figure 1A). Neither selective ß3-AR agonists (BRL37344 and CL316243 [GenBank] ) nor non-conventional partial agonists (CGP12177 and cyanopindolol, which activate ß3-AR and l.a.s.ß1-AR) elicited significant relaxation, up to 30 µM (Figure 1B).


Figure 1
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  		Figure 1 Effect of isoproterenol (A), CGP12177, cyanopindolol, CL316243, and BRL37344 (B) in mice extralobar pulmonary arteries. Relaxant response to isoproterenol was evaluated in the absence or presence of ICI118551 (ICI), CGP20712A (CGP), and SR59230A (SR) at indicated concentrations. * P < 0.05, *** P < 0.001 (ANOVA).

 
3.2. Contribution of endothelial NO and cAMP/PKA pathways to ß-AR-mediated relaxation of mice extralobar pulmonary arteries
L-NAME (300 µM) or endothelium removal decreased relaxation to isoproterenol in extralobar pulmonary arteries (Figure 2A). The L-NAME-insensitive relaxant component to isoproterenol was abolished by 0.1 µM CGP20712A (0.2 ± 3.6% relaxation at 3 µM isoproterenol, n = 3). In endothelium-denuded arteries, the response to isoproterenol was not modified by L-NAME (Figure 2A) or ICI118551, but it was abolished by CGP20712A (Figure 2B). As expected from its selectivity as ß2-AR agonist, procaterol elicited a relaxant effect, which was abolished by 0.1 µM ICI118551 but not modified by 0.1 µM CGP20712A (not shown). Procaterol-induced relaxation was abolished by L-NAME, even in the presence of a subthreshold concentration (30 nM) of the NO donor, DEA-NO, and also by ODQ (1 µM; an inhibitor of soluble guanylate cyclase) or endothelium removal (Figure 2C).


Figure 2
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  		Figure 2 Effect of isoproterenol (A and B) and procaterol (C and D) in extralobar pulmonary arteries with (ENDO+) or without (ENDO–) functional endothelium, isolated from wild-type (WT) or eNOS–/– mice. Relaxant response to isoproterenol was evaluated in the absence or presence of 300 µM L-NAME (A), 0.1 µM ICI118551 (ICI) or 0.1 µM CGP20712A (CGP) (B). Relaxant response to 0.1 µM procaterol was evaluated in the absence or presence of 300 µM L-NAME, 300 µM L-NAME + 30 nM DEA-NO, or 1 µM ODQ (C), or in eNOS–/– mice in the absence or presence of 300 µM L-NAME or 300 µM L-NAME + 10 µM indomethacin (indo) + 0.1 µM apamin + 0.1 µM charybdotoxin (ChTX) (D). A and B: * P < 0.05, ** P < 0.01, *** P < 0.001 (ANOVA). C and D: ** P < 0.01, *** P < 0.001 vs. procaterol control; # P < 0.05 vs. procaterol in eNOS–/– (Student's t-test).

 
In extralobar pulmonary arteries from eNOS–/– mice, relaxation to procaterol was decreased, but not abolished, compared to wild-type (Figure 2D). However, L-NAME failed to modify the effect of procaterol in arteries from eNOS–/– mice, while further addition of inhibitors of cyclooxygenases (10 µM indomethacin) and EDHF (0.1 µM charybdotoxin and 0.1 µM apamin) almost completely blunted it (Figure 2D). Relaxation to acetylcholine (as reference endothelial NO-dependent agent) was similarly diminished, but not abolished, in extralobar pulmonary arteries from eNOS–/– mice [10 µM acetylcholine inducing 62.4 ± 1.1% (n = 69) and 8.5 ± 2.3% relaxation (n = 17), in wild-type and eNOS–/– mice, respectively; P < 0.001]. As for procaterol, acetylcholine-induced relaxation in these arteries was unaffected by L-NAME (not shown), but abolished by further addition of indomethacin, charybdotoxin, and apamin (–2.2 ± 1.7% relaxation at 10 µM acetylcholine, n = 4).

Isoproterenol and procaterol responses were also evaluated in the presence of adenylate cyclase (SQ22536, 300 µM) or PKA (Rp-8-Br-cAMPS, 100 µM) inhibitors. Both drugs decreased the effect of isoproterenol in endothelium-denuded extralobar pulmonary arteries, but not the one of procaterol in arteries with endothelium (Figure 3).


Figure 3
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  		Figure 3 Effect of 300 µM SQ22536 or 100 µM Rp-8-Br-cAMPS on the relaxant responses to isoproterenol (A) and 0.1 µM procaterol (B) in mice extralobar pulmonary arteries with (B) or without (ENDO–; A) functional endothelium. ** P < 0.01 (ANOVA).

 
Immunostaining for ß1-AR was detected in both medial and endothelial layers of extralobar pulmonary artery (Figure 4A). However, ß2-AR was detected in the endothelial layer, but not in the medial layer (Figure 4B).


Figure 4
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  		Figure 4 Immunostaining for ß1-AR (A) and ß2-AR (B) in sections of mice extralobar pulmonary arteries. Pictures are representative of two independent experiments. Magnifying objective x1000. e indicates endothelial layer staining; m indicates medial layer staining.

 
3.3 Characterization of ß-AR subtypes and contribution of endothelial NO to ß-AR-mediated relaxation of mice intralobar pulmonary arteries
In intralobar pulmonary segments, isoproterenol elicited a relaxation, which was reduced by L-NAME (Figure 5A). Procaterol also induced a relaxant effect (ICI118551-sensitive but CGP20712A-insensitive, not shown), which was suppressed by L-NAME (Figure 5B). As in extralobar segments, procaterol- (Figure 5B) and acetylcholine (not shown)-induced relaxations were diminished, but not abolished, in intralobar pulmonary arteries from eNOS–/– mice. These residual relaxations to both procaterol (Figure 5B) and acetylcholine were insensitive to L-NAME [10 µM acetylcholine inducing 19.4 ± 2.7% (n = 8) and 18.7 ± 6.4% relaxation (n = 6), in the absence or presence of L-NAME, respectively]. As in extralobar segments, neither cyanopindolol nor CL316243 [GenBank] did modify the contractile tone in intralobar pulmonary arteries (Figure 5C).


Figure 5
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  		Figure 5 Effect of isoproterenol (A), procaterol (B), cyanopindolol, or CL316243 (C) in mice intralobar pulmonary arteries. Relaxant responses to isoproterenol were evaluated in the absence or presence of 300 µM L-NAME (A). Relaxant responses to 0.1 µM procaterol were evaluated in wild-type (WT) or eNOS–/– mice in the absence or presence of 300 µM L-NAME (B). A: * P < 0.05 (ANOVA). B: ** P < 0.01 vs. procaterol WT (Student's t-test).

 
3.4 Effects of chronic hypoxia on endothelial NO-dependent relaxation in mice extralobar and intralobar pulmonary arteries
Chronic hypoxia induced a significant rise in the weight ratio (right ventricle/left ventricle+septum) and in the mean right ventricular pressure (Table 1).


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  		Table 1 Right ventricule/(left ventricule+septum) weight ratio and mean right ventricular pressure in mice exposed to normoxia or chronic hypoxia

 
In extralobar pulmonary arteries from hypoxic mice, relaxations to isoproterenol and procaterol were similar to that obtained in control mice (Figure 6A and B), and endothelium removal reduced the responses to these ß-AR agonists to the same extent as in control mice (Figure 6A and B). In these arteries, relaxation to acetylcholine (10 µM), but not to SNP (1 µM, a NO donor), were markedly decreased, in comparison to the responses obtained in extralobar pulmonary arteries from control mice (Figure 6B).


Figure 6
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  		Figure 6 Effect of isoproterenol (A and C), 0.1 µM procaterol (Proc.), 10 µM acetylcholine (Ach) or 1 µM SNP (B and D) in extralobar (A and B) and intralobar (C and D) pulmonary arteries from normoxic (NX) or hypoxic (CH) mice. Relaxant responses to isoproterenol and procaterol were evaluated in arteries with (ENDO+) or without (ENDO–) functional endothelium (A and B), or in the absence or presence of 300 µM L-NAME (C and D). A and C: * P < 0.05, ** P < 0.01, *** P < 0.001 (ANOVA). B and D: ** P < 0.01 vs. ENDO+; ## P < 0.01, ### P < 0.001 vs. normoxic; $ P < 0.05, $$$ P < 0.001 vs. procaterol (Student's t-test).

 
In intralobar pulmonary arteries from hypoxic mice, the relaxant responses to isoproterenol, procaterol, and SNP were all increased in comparison to those observed in control mice (Figure 6C and D), while relaxation to acetylcholine was significantly reduced (Figure 6D). In these arteries, relaxant responses to isoproterenol and procaterol were inhibited by L-NAME, in a similar way to that in control mice (Figure 6C and D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The main findings of this study are that, in mice extra- and intra-lobar pulmonary arteries: (i) both ß1- and ß2-, but not ß3-AR, mediate relaxation; (ii) ß2-AR are located in the endothelial cells, and mediate endothelial NO-dependent relaxation; (iii) this ß2-AR relaxation is preserved following chronic hypoxia, in contrast to the endothelial NO-dependent relaxation to acetylcholine.

The potential relaxant role of ß3-AR and l.a.s.ß1-AR in mice pulmonary circulation has never been specifically assessed. The present study does not provide pharmacological evidence for a relaxant role of these receptors in mice extra- and intra-pulmonary arteries. The ß3-AR antagonist SR59230A did not induce further inhibition of isoproterenol-induced relaxation in the presence of ß1- and ß2-AR antagonists (the potential antagonist activity of SR59230A on ß1- and ß2-AR22 being minimized under these conditions). Moreover, increasing concentration of CGP20712A from 0.1 to 3 µM for blockade of l.a.s.ß1-AR23 did not induce larger inhibition of isoproterenol-induced relaxation. Finally, agonists of ß3- and l.a.s.ß1-AR (CGP12177, cyanopindolol), as well as more selective ß3-AR agonists (BRL37344, CL316243 [GenBank] ) failed to induce relaxation.

In accordance with a previous report,17 this study demonstrates the relaxant role of ß1-AR in mice pulmonary arteries. Indeed, relaxation to isoproterenol was almost abolished by the ß1-AR antagonist (CGP20712A) in endothelium-denuded arteries. Consistently, immunohistochemistry demonstrated localization of ß1-AR in the medial layer of pulmonary arteries. The ß1-AR-dependent relaxation was also inhibited by adenylate cyclase or PKA inhibitors. Thus, according to the Gs coupling of the receptor,24 activation of ß1-AR located on smooth muscle cells elicits relaxation through activation of the cAMP/PKA pathway in mice pulmonary arteries.

New findings of the present study are the relaxant role of ß2-AR and the endothelial expression of this receptor in mice pulmonary arteries. While it was ineffective in pulmonary arteries without endothelium, the ß2-AR antagonist ICI118551 antagonized the effect of isoproterenol in pulmonary artery with endothelium. Moreover, the selective ß2-AR agonist procaterol elicited marked relaxant response only in arteries with endothelium. Immunohistochemistry provides direct evidence for localization of ß2-AR in the endothelial layer, but not in the medial layer of pulmonary arteries. In addition to ß2-AR, ß1-AR was also found in the endothelial layer of pulmonary arteries, and probably contributed to the effect of the non-selective ß-AR agonist, isoproterenol. Thus, not only ß1-AR, but also ß2-AR, mediates relaxation in mice pulmonary arteries, the latter component being endothelium-dependent. These findings contrast with those obtained in ß1- and/or ß2-AR knockout mice, in which ß-AR-induced relaxation of pulmonary artery was exclusively dependent on ß1-AR.17 However, in contrast to the present one, no evidence was provided in the latter study for endothelial functionality in the studied pulmonary arteries.

The present study also demonstrates that the endothelial ß2-AR elicits NO-dependent relaxation. Indeed, isoproterenol-induced relaxation was partially inhibited by L-NAME or eNOS gene deletion, whereas procaterol-induced relaxation was almost abolished under these conditions, or in the presence of ODQ, an inhibitor of guanylate cyclase activation by NO. The eNOS activity underlying relaxation seems exclusively located on endothelial cells, since L-NAME failed to inhibit the effect of isoproterenol in endothelium-denuded preparations. Inhibition of isoproterenol-induced relaxation was greater in the presence of L-NAME than after endothelium removal. This appears unlikely due to incomplete removal of endothelial cells by CHAPS (since acetylcholine failed to induce relaxation in the same endothelium-denuded arteries), but might be explained by the release of endothelium-derived contracting factors, counteracting relaxation to isoproterenol. Nevertheless, while previous studies17 did not specifically address the endothelium- and eNOS-dependencies of ß-AR-mediated relaxation in mice pulmonary artery, the present data clearly demonstrate the involvement of two components, a ß2-AR/endothelium/eNOS-dependent one and a ß1-AR-dependent but endothelium/eNOS-independent one, in both extra- and intra-lobar pulmonary arteries. Either stimulation of NO production by eNOS or permissive role of basal eNOS-derived NO21 may account for the endothelium/eNOS dependency of ß2-AR-mediated relaxation. Since procaterol failed to induce relaxation in the presence of L-NAME and a subthreshold concentration of the NO donor DEA-NO, it rather appears that endothelial ß2-AR stimulates NO production by eNOS. Male (as used in this study) and female mice may differ in the relative contribution of NO and other relaxing factors to endothelium-dependent vasorelaxation in systemic resistance arteries.25 Whether pulmonary arteries and endothelium-dependent ß2-AR relaxation also display such differences needs further investigation. In contrast to data obtained in systemic arteries,9 the NO-dependent ß2-AR-mediated relaxation of mice pulmonary arteries was not mediated by the cAMP/PKA pathway, since neither adenylyl cyclase, nor PKA inhibitors did modify procaterol-induced relaxation. The ß2-AR-mediated eNOS stimulation might result from activation of Gi/o/phosphoinositide 3-kinases (PI3K)/Akt pathway,24,26 a well-known stimulus of eNOS.27 Mechanisms underlying the ß2-AR-mediated eNOS activation in mice pulmonary arteries require further characterization.

Unexpectedly, procaterol-induced relaxation (as well as acetylcholine-induced one) was only partly diminished in pulmonary arteries from eNOS–/– mice. Since residual relaxation to procaterol (and to acetylcholine) in arteries from eNOS–/– mice was L-NAME-insensitive, but inhibited by cyclooxygenases and EDHF inhibitors, genetic deletion of eNOS likely promotes a switch of endothelial ß2-AR (and also muscarinic receptor) coupling from eNOS to cyclooxygenases- and/or EDHF-dependent relaxant pathways. Preservation of an endothelium-dependent relaxation in eNOS–/– mice has been already reported in systemic circulation,25,28 but not, to our knowledge, in the pulmonary artery nor for ß-AR-mediated relaxation. Switch from NO to other pulmonary vasorelaxant factors might partly explain why eNOS–/– mice used here did not develop pulmonary arterial hypertension, as indicated by the lack of right ventricular pressure elevation. Other potential compensatory mechanisms following eNOS gene deletion include activation of atrial natriuretic peptide system29 or expression of inducible NOS.30 The latter appears unlikely, since this isoform was not immuno-detected in pulmonary arteries from eNOS–/– mice (not shown). It is noteworthy that some contradictory results on the influence of eNOS deletion appear to depend on the genetic background, since pulmonary hypertension was observed in mixed C57BL6/sv129 eNOS–/– mice,31,32 but not in those from C57BL633 as used in the present study.

Another unexpected finding of the present study is that the endothelial NO-dependent ß2-AR relaxation is preserved following chronic hypoxia. Pulmonary hypertension was proved in hypoxic mice by increasing right ventricular pressure and weight. In accordance with a previous study,18 endothelial dysfunction was demonstrated in both extra- and intra-lobar pulmonary arteries from hypoxic mice by a decrease in the relaxant effect of acetylcholine, while relaxation to the NO donor SNP was either not affected (extralobar segments) or even increased (intralobar segments). It is shown here that extralobar pulmonary arteries from hypoxic mice exhibited very similar ß-AR relaxant responses and sensitivity to NOS inhibition or endothelium removal to those in arteries from control mice. In intralobar pulmonary arteries, while the L-NAME-insensitive relaxation to isoproterenol was not modified following chronic hypoxia, the effect of isoproterenol in the absence of L-NAME was enhanced. This suggests that the NO-dependent component of isoproterenol-induced relaxation, i.e. the ß2-AR-mediated one, was increased in intralobar pulmonary arteries from hypoxic mice. Accordingly, the relaxant effect of procaterol was also enhanced in these arteries, as well as relaxation to the exogenous NO donor, SNP. Such enhancement of NO-dependent relaxation may result from hypoxia-induced up-regulation of soluble guanylate cyclase or cGMP-dependent protein kinase expression, and vascular smooth muscle calcium desensitization through inhibition of RhoA/Rho kinase.34–36

We have previously shown that pulmonary arteries from hypoxic mice exhibited an increased level of reactive oxygen species (ROS, including superoxide anions) in all vascular layers.18 Thus, despite ROS elevation, NO-dependent relaxation mediated by ß2-AR was not diminished in pulmonary arteries from hypoxic mice. This further supports the idea that in these arteries, ROS are not direct mediators of decreased NO bioavailability.18 This might be explained by differential subcellular localization of superoxide anion and NO production, or by a major contribution of hydrogen peroxide (which does not directly inactivate NO, contrary to superoxide anions) in ROS elevation.18 A major finding of the present study is that endothelial NO-dependent relaxation to ß2-AR agonists was preserved in pulmonary arteries from hypoxic mice, while endothelial NO-dependent relaxation to acetylcholine was diminished. As discussed above, endothelial ß2-AR can activate eNOS by mechanisms (PI3K/Akt pathway, for instance), which are likely distinct from those responsible for eNOS activation by Gq/11-coupled muscarinic receptors. Thus, in pulmonary arteries, endothelial NO-dependent relaxations may be affected or not by chronic hypoxia, depending on the mechanisms underlying eNOS activation. Interestingly, differential alterations of eNOS-mediated relaxations have been also recently found in peripheral vasculature.37,38 For instance, chronic heart failure almost abolished flow-mediated dilatation, without markedly affecting the response to acetylcholine.37

In conclusion, both ß1- and ß2-AR, but not ß3- or l.a.s.ß1-AR, mediate relaxation of mice extra- and intra-lobar pulmonary arteries. The endothelium-independent relaxation is mediated by ß1-AR, while the ß2-AR component is endothelium- and eNOS-dependent. Furthermore, in pulmonary arteries from hypoxic mice, which exhibit decreased endothelium-dependent relaxation to acetylcholine, the endothelial NO-dependent relaxation mediated by ß2-AR is preserved, suggesting that the mechanisms underlying eNOS activation are determinant for expression of hypoxia-induced endothelial dysfunction in pulmonary arteries. Interestingly, a recent pilot trial demonstrates the beneficial effects of ß2-AR agonists on pulmonary haemodynamics in patients with chronic obstructive pulmonary disease, as a result of a direct vasodilator effect on pulmonary vessels.39 These data highlight the role of ß2-AR as pharmacological target to decrease pulmonary arterial resistance and to exert other endothelial NO-related protective effects in hypoxic pulmonary vasculature.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was partially supported by grants from Conseil Régional d'Aquitaine (20030301301A), Fondation de France (2004002928 and 2006005603) and ANR (Physio, HPV-PAH in COPD).


   Acknowledgements
 
The authors would like to thank Mrs Lacayrerie and Mr Guignard for excellent animal care, Mr Techoueyres for maintenance of hypobaric chamber, and Mrs Barbier, Castoreo, Leguelinel, Martin Vila, and Papon for their technical contribution to this work.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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E. Delannoy, A. Courtois, V. Freund-Michel, V. Leblais, R. Marthan, and B. Muller
Hypoxia-induced hyperreactivity of pulmonary arteries: role of cyclooxygenase-2, isoprostanes, and thromboxane receptors
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