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BOR - Papers in Press, published online ahead of print September 21, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043695
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BIOLOGY OF REPRODUCTION 74, 209–216 (2006)
DOI: 10.1095/biolreprod.105.043695
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Stimulation of the ADRB3 Adrenergic Receptor Induces Relaxation of Human Placental Arteries: Influence of Preeclampsia1

C. Rouget 345  , O. Barthez 3, F. Goirand 3, M.J. Leroy 5, M. Breuiller-Fouché 5, Z. Rakotoniaina 3, P. Guérard 3, E.J. Morcillo 6, C. Advenier 4, P. Sagot 7, D. Cabrol 58 , M. Dumas 3, and M. Bardou 2 34 

Laboratory of Cardiovascular Physiopathology and Pharmacology,3 Faculty of Medicine, 21079 Dijon Cedex, France UPRES EA220-Pharmacology,4 UFR Biomédicale des Saints Pères, 75006 Paris, France INSERM U427,5 Faculté de Pharmacie, 75006 Paris, France Department of Pharmacology,6 University of Valencia, 46010 Valencia, Spain Department of Gynaecology,7 CHU du Bocage, 21000 Dijon, France Department of Obstetrics and Gynecology,8 Port-Royal Cochin Hospital, 75014 Paris, France

ABSTRACT

Preeclampsia, which complicates 3–8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality. Its pathophysiology remains unclear. The aim of the present study was to investigate the presence and the role of ß2- and ß3-adrenergic receptors (ADRB2 and ADRB3, respectively) in human placental arteries and to assess the influence of preeclampsia on ADRB responsiveness. SR 59119A, salbutamol, and isoproterenol (ADRB3, ADRB2, and nonselective ADRB agonists, respectively) induced a concentration-dependent relaxation of placental artery rings obtained from women with uncomplicated or preeclamptic pregnancies. SR 59119A-induced relaxation was unaffected by the blockade of ADRB1 and ADRB2 by 0.1 µM propranolol but was significantly decreased by the blockade of ADRB1, ADRB2, and ADRB3 by 10 µM propranolol. Both SR 59119A and salbutamol were associated with a significant increase in cAMP production that was significantly inhibited by pretreatment with 0.1 µM propranolol only for salbutamol. SR 59119A-induced relaxation (Emax = 28% ± 5% vs. 45% ± 4%, respectively) and cAMP production (2.7 ± 0.5 vs. 4.9 ± 0.4 pmol/mg of protein, respectively; P < 0.01) were decreased in arteries obtained from preeclamptic compared to normotensive women. Both ADRB2 and ADRB3 transcripts were expressed at the same level between arteries from normotensive and preeclamptic women. Western blot analysis, however, revealed a decreased expression of the ADRB3 immunoreactive protein in arteries from preeclamptic compared to normotensive women. We suggest the presence of functional ADRB2 and ADRB3 in human placental arteries. Even if preeclampsia is associated with an impairment of the ADRB3 responsiveness, ADRB3 agonists may have future pharmaceutical implications in the management of pregnancy-related disorders.

catecholamines, cyclic adenosine monophosphate, cyclic guanosine monophosphate, placenta, pregnancy

INTRODUCTION

Preeclampsia, which complicates 3–8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality and is responsible for one-third of maternal deaths [1]. Several studies have suggested that women who develop preeclampsia are at increased risk of cardiovascular complications [2]. The mechanisms responsible for this pathology are still unclear. It is characterized by abnormal vascular response to placentation associated with increased systemic vascular resistance, enhanced platelet aggregation, activation of the coagulation system, and endothelial cell dysfunction [3]. The initiating event of preeclampsia seems to be the reduction of uteroplacental perfusion associated with an endothelial activation, which leads to enhanced formation of endothelin-1 and thromboxane A2, decreased synthesis of vasodilatators (e.g., nitric oxide [NO]), and finally, to maternal hypertension [4]. Furthermore, it has been suggested that fetoplacental resistances are increased during preeclampsia [5]. Among the multiple theories regarding the pathophysiology of preeclampsia (for review, see Sibai et al. [6]), it recently was speculated that an imbalance of pro- and antiangiogenic factors may lead to preeclampsia [7, 8]. Trophoblasts have the capacity to secrete catecholamines, and high venous blood concentrations of noradrenaline were found in preeclamptic pregnancies [9]. It can be postulated (even if not unanimously accepted [10]) that the high levels of catecholamines found in the plasma of women with preeclampsia might be of placental origin, in response to trophoblast tissue ischemia from preeclampsia, and secreted as a physiological signal to increase maternal blood flow to the fetoplacental unit. Little is known, however, about the role of the ß-adrenergic receptor (ADRB) during preeclampsia. Aune et al. [10] have shown, using a human mononuclear leukocytes model, that the number of functional ADRB2 is reduced in preeclampsia without increased plasma catecholamine levels, suggesting that the reduction of ADRB2 could be one of the factors implicated in the increase of peripheral vascular resistances.

Although the ADRBs originally were subclassified into ADRB1 and ADRB2, another subtype, the ß3 subtype, has been reported [11]. The ADRB3 shares 40–50% amino acid sequence identity with ADRB1 and ADRB2 [12], and it lacks recognition sites for the cAMP-dependent protein kinase and ADRB kinase implicated in the desensitization of ADRB2 [13]. The ADRB3 has been shown to mediate lipolysis in white adipose tissue and thermogenesis in brown adipose tissue [14], to inhibit the contractile activity of colon [15], and to inhibit in vitro contraction of human near-term or nonpregnant myometrium [16, 17].

The vasorelaxant effect of ADRB3 agonists has been demonstrated in several animal models. For example, Berlan et al. [18] demonstrated that ADRB3 is present in dog cutaneous microvessels and that its in vivo stimulation by selective agonists induces vasorelaxation, as suggested by the increase of cutaneous blood flow. The vasorelaxant effect of the ADRB3 agonists was described in rat thoracic aorta [19]. On the other hand, little is known about the presence and role of ADRB3 in the human cardiovascular system. Furthermore, transduction mechanisms of this receptor are still misunderstood. Classically, the ADRB3 is coupled to Gs protein and stimulates the adenylyl cyclase/cAMP pathway [20], as is the case in human myometrium [16, 17, 21]. Nevertheless, Gauthier et al. [22] in human heart and Trochu et al. [19] in rat thoracic aorta have shown that ADRB3 agonists act through a NO synthase pathway, leading to an increase in cGMP level.

To our knowledge, the presence of the ADRB2 and ADRB3 in human placental arteries has not been described convincingly, even if it was recently suggested that ADRB3 might be present in human umbilical arteries [23]. We, therefore, investigated the presence and function of ADRB2 and ADRB3 in human placental arteries and assessed the influence of preeclampsia on their expression and function using complementary functional, biochemical, and molecular approaches.

MATERIALS AND METHODS

Preparation of Human Placental Arteries

Placental arteries were dissected from the chorionic plate of placentae obtained from women with uncomplicated (n = 45) or preeclamptic (n = 18) pregnancies at term (mean gestation, 38.5 ± 0.31 and 37.6 ± 1.26 wk for normotensive and preeclamptic pregnancies, respectively), immediately after vaginal delivery or cesarean section. The diagnostic criteria used to select preeclamptic women for the present study were as follows: New-onset hypertension was defined as a blood pressure of at least 140 mm Hg (systolic) or at least 90 mm Hg (diastolic) on at least two occasions and at least 4–6 h apart after the 20th week of gestation in women known to be normotensive beforehand, and proteinuria was defined as excretion of 300 mg or more of protein every 24 h. If 24-h urine samples are not available, proteinuria is defined as a protein concentration of 300 mg/L or more (≥1 on dipstick) in at least two random urine samples taken at least 4–6 h apart [6]. No significant difference was observed in vasoreactivity of arteries obtained from vaginal delivery or cesarean section. Among women with preeclampsia, one received a treatment with intravenous nicardipine (a calcium-channel antagonist) and another with magnesium sulfate. None of the patients had been treated with ADRB2 agonist before delivery. Placental arteries were placed immediately in ice-cold Krebs solution (116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.6 mM KH2PO4, 0.8 mM MgSO4, 19 mM NaHCO3, 5.5 mM glucose, and 0.1 mM L-ascorbic acid; 4°C) and transported to the laboratory to be used fresh (functional and biochemical studies) or quickly frozen in liquid nitrogen before being stored at –80°C until use (molecular biology and Western blot experiments). The use of human placental arteries for experiments was approved by the local ethical committee.

Functional Study

Arteries were cut into rings (length, 5 mm; diameter, 1–2 mm) and were suspended isometrically between two stainless-steel hooks under an optimal resting tension of 3 g [5] in a 10-ml organ bath containing Krebs solution (composition as described above) at 37°C and continuously gassed with a mixture of 95% oxygen and 5% carbon dioxide (pH 7.40). The arterial rings were attached to a force-displacement transducer connected to an amplifier (EMKA) to record tension changes by a graphics software IOX (EMKA). After 1 h, during which the arterial rings were washed every 15 min and the resting tension readjusted to 3 g, the tissues were allowed to equilibrate for 30 min before being challenged with 80 mM potassium chloride to evaluate their functional integrity. Arteries were then washed three times at 15-min intervals and then allowed to equilibrate for a further 30 min. After recovery, arterial rings were contracted with 10 nM U 46619, a thromboxane A2 analog. When the developed tension had reached a plateau, cumulative concentration-response curves (CRCs; 0.001–10 µM) were constructed for isoproterenol (a nonselective ADRB agonist), salbutamol (an ADRB2 agonist), and SR 59119A (an ADRB3 agonist). Each following concentration was added to the bath when a plateau of relaxation was reached (~20 min). One additional paired ring was used as a control for any time-related change in the tension throughout the experimental procedure. A second set of experiments was performed during which CRCs were constructed after a 30-min incubation with propranolol at 0.1 µM to block ADRB1 and ADRB2 or at 10 µM to block ADRB1, ADRB2, and ADRB3 [24] or with L-NNA (N-{omega}-nitro-L-arginine methyl ester, 100 µM) to block NO synthase. Finally, to assess the role of endothelium in the effects of SR 59119A, a third set of experiments was performed in which the relaxing properties of SR 59119A were assessed in a paired fashion in endothelium-intact or endothelium-denuded placental artery rings. Endothelium was denuded mechanically. The Emax value indicates the maximal relaxation obtained at the maximal concentration tested (10 µM) for each ADRB agonist and is expressed as a percentage of the maximal relaxation obtained with 0.1 mM papaverine added to the bath at the end of each experiment. The EC50 values indicate the concentration producing 50% of maximal relaxation induced by each drug and are calculated using the GraphPad Prism 4.01 computer program (GraphPad Software). For analysis, EC50 values were log-transformed and expressed as –log EC50.

Biochemical Study

Arterial rings were equilibrated in Krebs solution (composition as described above) continuously gassed with a mixture of 95% oxygen and 5% carbon dioxide at 37°C (pH 7.40) for 40 min and then exposed to 0.1 µM propranolol to block ADRB1 and ADRB2 [24] or its saline vehicle for 30 min, followed by addition of ADRB agonists (salbutamol or SR 59119A; both 10 µM) for 5 min. In paired arterial rings, time-matched experiments were carried out for a total incubation period of 65 min to determine the basal cyclic nucleotide content of nonstimulated tissues. At the end of the incubation period, the tissues were processed as described previously [21]. Arterial rings were frozen immediately in liquid nitrogen and homogenized in ice-cold 10% trichloroacetic acid. The homogenate was centrifuged at 10000 x g for 15 min at 4°C. The pH of the supernatant was neutralized by the addition of excess calcium carbonate followed by low-speed centrifugation. Aliquots of the supernatant were tested for cAMP and cGMP by enzyme immunoassay kits (RPN 225 and RPN 226, respectively; Amersham Pharmacia Biotech Ltd.) according to the manufacturer's instructions.

Analysis of ADRB2 and ADRB3 Transcripts by RT-PCR

As described previously [17, 21, 25], extraction of total RNA from placental arteries using the Trizol reagent method, RT using Moloney murine leukemia virus reverse transcriptase, and PCR reactions using Taq DNA polymerase were performed under the conditions recommended by the manufacturer (Life Technologies).

The design of primers for each ADRB (Invitrogen custom primers) and experimental procedures were exactly as described in previous reports [17, 21]. Briefly, the amplification profile for ADRB2 and ADRB3 transcripts consisted in denaturation at 94°C for 1 min, annealing at 64°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. As a positive control for the assay, reverse-transcribed ADRB2 and ADRB3 mRNAs were amplified by PCR in human myometrium, which is known to express these receptors [17, 21, 26]. We controlled so that PCR products without previous reverse transcriptase did not reveal any positive band.

For semiquantification, the intensities of the bands on Polaroid pictures of the ethidium bromide staining gel were analyzed densitometrically using the image-analyzer Gel Doc 1000 system (Bio-Rad). Results are expressed as relative levels (arbitrary densitometric units [ADU]) of specific mRNA normalized to ß2-microglobulin mRNA in each sample.

Western Blot Analysis

Western blot experiments were performed following the same experimental procedure as reported previously [17]. Briefly, snap-frozen placental arteries obtained from normotensive and hypertensive pregnancies were homogenized with Ultra-Turrax in homogenization buffer. After an initial centrifugation (500 x g for 15 min at 4°C), the supernatant was removed and centrifuged at 48000 x g for 20 min at 4°C. The resulting pellet was suspended in solubilization buffer overnight at 4°C. A further centrifugation was performed at 48000 x g for 20 min at 4°C. Total protein content was determined by the Bradford method with BSA as standard. Samples (50 µg of protein by lane) were dissolved in Laemmli buffer (4x) and boiled for 5 min before electrophoresis on a 10% SDS-PAGE. Then, proteins were transferred to a nitrocellulose membrane. Blots were blocked for 1 h in 10% nonfat dried milk powder in TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20; pH 7.8) at room temperature. Blocked membranes were washed three times with TBST. The blots were then incubated overnight at 4°C with a 1:500 dilution of primary ADRB3 polyclonal antibody (AB5122; Chemicon International) in 1% nonfat dried milk powder in TBST. After three washes with TBST, the blots were incubated for 45 min with horseradish peroxidase-conjugated anti-rabbit IgG whole antibody (NA 934; Amersham) at a dilution of 1:5000 at room temperature and washed 5 times with TBST. Immunoreactive proteins were detected by chemiluminescence (ECL detection reagents; RPN2105; Amersham Pharmacia Biotech). The intensities of the bands were analyzed densitometrically by the NIH software package and expressed in ADUs as the mean ± SEM. The specificity of each immunoreactive band was assessed by specific blocking in the presence of the antigenic peptide against which the antibody has been raised (AG388; Chemicon International). For preabsorption, a mixture of the primary antibody with its respective antigen (1:5) was incubated under mild agitation in a small volume of PBS buffer for 24 h at 4°C. In addition, homogenate of Chinese hamster ovary (CHO) cells transfected with the human ADRB3 (graciously provided by Dr. P. Marini from Sanofi-Aventis Research Center, Milan, Italy) was used as positive control.

Histological Evaluation of Placental Arteries

To compare morphology of placental arteries and to assess endothelial integrity (agents inducing endothelium-dependent vasorelaxation [e.g., acetylcholine, adenosine diphosphate, and bradykinin] usually fail to relax placental arteries [27]), histological evaluation of arteries was performed at the end of experiments (standard coloration by hematoxylin-eosin-safran and immunostaining using a monoclonal antibody against CD31 [DAKO]).

Drugs and Solutions

The drugs and chemicals used and their sources were as follows: SR 59119A (N-[(7-methoxy-1,2,3,4-tetrahydronaphthahlen-(2R)-2-yl)methyl]-(2R)-2-hydroxy-2-(3-chlorophenyl)ethanamine hydrochloride; a gift from Sanofi-Aventis Research Center, Milan, Italy), and U-46619 (9,11-dideoxy-11{alpha},9{alpha}-epoxymethano-prostaglandin F2{alpha}), L-NNA, serotonin, papaverine, salbutamol, isoproterenol, and propranolol (Sigma-Aldrich Chimie). SR 59119A was dissolved in a mixture of absolute ethanol (30%), dimethyl sulfoxide (2%), and distilled water for the 10 µM solution and, thereafter, diluted in distilled water, whereas all other drugs were dissolved in distilled water. Drug concentrations are given as final bath concentrations.

Statistical Analysis

In the functional experiments, differences among groups were determined by ANOVA and expressed with a t-test using the Bonferroni correction. Differences in potencies and maximal effects, as well as differences among groups in biochemical experiments, were determined by Student t-test for unpaired data or ANOVA as appropriate. All differences were considered to be significant at P < 0.05.

RESULTS

Functional Study

SR 59119A, salbutamol, and isoproterenol induced a concentration-dependent relaxation of U46619-contracted placental artery rings obtained from women with uncomplicated pregnancy (Fig. 1A). The maximal effect obtained for 10 µM SR 59119A was significantly (P < 0.01) higher than that obtained for isoproterenol and salbutamol at the same maximal concentration (10 µM) (Table 1). The –log EC50 value for SR 59119A was not significantly superior to that of salbutamol and isoproterenol (Table 1). The relaxing properties of SR 59119A was not significantly reduced in endothelium-denuded compared with endothelium-intact placental artery rings (Emax, 45% ± 3% vs. 39% ± 3%, respectively; –log EC50, 7.1 ± 0.30 vs. 6.89 ± 0.29, respectively) (Fig. 1B).


Figure 1
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  		FIG. 1. A) Effect of salbutamol, SR 59119A, and isoproterenol (ADRB2, ADRB3, and nonselective ADRB agonists, respectively) on U46619 (10 nM)-contracted placental arteries obtained from normotensive women. Results are expressed as the mean ± SEM (n = 7–20 arteries, each artery obtained from a single woman). B) Relaxing properties of SR 59119A in endothelium-intact or endothelium-denuded placental artery rings. Results are expressed as the mean ± SEM (n = 7, with each artery obtained from a single woman)


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  		TABLE 1. Emax and -log EC50 values for isoproterenol, salbutamol, and SR 59119A in human placental arteries

The SR 59119A CRC was significantly (P < 0.001) downward-shifted after the blockade of ADRB1, ADRB2, and ADRB3 with 10 µM propranolol but was unaffected by the blockade of ADRB1 and ADRB2 with 0.1 µM propranolol pretreatment (Fig. 2A and Table 2).


Figure 2
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  		FIG. 2. A) Effect of propranolol pretreatment (0.1 or 10 µM) on the concentration-response curve for SR 59119A in human placental artery rings obtained after uncomplicated pregnancies. Results are expressed as the mean ± SEM (n = 12 arteries, with each artery obtained from a single woman). B) Effect of L-NNA pretreatment (100 µM) on the concentration-response curve for SR 59119A in human placental artery rings obtained after uncomplicated pregnancies. Results are expressed as the mean ± SEM (n = 10 arteries, with each artery obtained from a single woman)


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  		TABLE 2. Emax and -log EC50 values for SR 59119A alone or in presence of propranolol 0.1 µM or 10 µM.a

The blockade of NO synthase by 100 µM L-NNA did not modify the CRC for SR 59119A (Emax, 45% ± 4% vs. 39% ± 3%, respectively; –log EC50, 6.81 ± 0.21 vs. 6.62 ± 0.23, respectively) in experiments performed with or without L-NNA (Fig. 2B).

Compared with arteries from normotensive women, the CRC for SR 59119A was downward-shifted (ANOVA, P < 0.05) in arteries obtained from preeclamptic women, with a significant reduction of maximal relaxation (Emax, 48% ± 4% vs. 28 ± 4% for 0.1 µM SR 59119A in arteries obtained from normotensive and preeclamptic women, respectively), whereas salbutamol- and isoproterenol-induced relaxations were not significantly different in both types of arteries (Fig. 3).


Figure 3
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  		FIG. 3. Influence of preeclampsia on the relaxing effects of SR 59119A, isoproterenol, and salbutamol on human placental arteries contracted with U46619 (10 nM). Results are expressed as the mean ± SEM (n = 7–20 arteries for normotensive and 6–8 for preeclamptic pregnancies, with each artery obtained from a single woman)

Cyclic Nucleotides Levels after Stimulation with Salbutamol and SR 59119A

SR 59119A and salbutamol (both 10 µM) induced a significant (P < 0.01) and similar increase in cAMP production compared to the basal value (Fig. 4 and Table 3), whereas they did not modify the cGMP level (data not shown). SR 59119A-induced cAMP production was not altered after pretreatment with 0.1 µM propranolol, whereas salbutamol effect was abolished by this treatment (Fig. 4 and Table 3).


Figure 4
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  		FIG. 4. Effects of salbutamol and SR 59119A (both at 10 µM) on cAMP production in placental arteries from normotensive women (n = 7) in the absence or presence of propranolol (0.1 µM). Results (mean ± SEM) are expressed in pmol/mg of protein. *P < 0.05 vs. basal production, ¥P < 0.05 vs. experiments performed in the absence of propranolol


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  		TABLE 3. Agonist-induced cAMP production in placental arteries obtained from normotensive women

Preeclampsia was associated with a significant impairment of SR 59119A-mediated cAMP production (2.7 ± 0.5 vs. 4.9 ± 0.4 pmol/mg of protein in arteries from preeclamptic and normotensive women, respectively; P < 0.01) and, to a lesser extent, of salbutamol-mediated cAMP production (Fig. 5).


Figure 5
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  		FIG. 5. Influence of preeclampsia on human placental arteries cAMP production (pmol/mg of protein) induced by salbutamol and SR 59119A (both at 10 µM). Results are expressed as the mean ± SEM. *P < 0.01 vs. normotensive arteries (n = 7 arteries for both normotensive and preeclamptic women, with each artery obtained from a single woman)

Analysis of ADRB2 and ADRB3 Transcripts

Using an RT-PCR method, we analyzed the expression of ADRB2 and ADRB3 transcripts in placental arteries obtained from normotensive and preeclamptic women. Both ADRB2 and ADRB3 mRNA expression was detected (417- and 368-bp fragments, respectively) in human placental arteries (Fig. 6A) and myometrium. Preeclampsia did not influence the level of expression of ADRB2 and ADRB3 transcripts (Fig. 6B).


Figure 6
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  		FIG. 6. A) Representative agarose gel electrophoresis of RT-PCR products of ADRB2 and ADRB3 in placental arteries from normotensive women (lanes a and b) or in human near-term myometrium (lanes c and d) used as positive control. Lanes b and d are without reverse transcription. Lane e corresponds to the DNA size marker.B) Semiquantification of ADRB2 and ADRB3 transcript levels of expression in placental arteries from six normotensive and six preeclamptic women. Results, expressed as relative levels in ADU, are presented as the mean ± SEM of specific mRNA normalized to ß2-microglobulin mRNA in each sample

Expression of ADRB3 Immunoreactive Protein

To characterize further the alteration of the ADRB3 function revealed in our functional study, we compared the expression of the ADRB3 immunoreactive protein in placental arteries from normotensive and preeclamptic women (Fig. 7A). Western blot analysis of plasma membranes prepared from arteries obtained from normotensive and preeclamptic pregnancies revealed a 68-kDa band [17], which disappeared in the presence of the corresponding blocking peptide (data not shown). As a positive control, a 68-kDa band was detected with a homogenate of CHO cells transfected with the human ADRB3 that also was specifically blocked by the antigenic peptide. Densitometric immunoblot analysis indicated a decreased intensity of the signal for ADRB3 in the placental arteries from preeclamptic compared to normotensive women (Fig. 7B).


Figure 7
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  		FIG. 7. A) Representative Western blot analysis of ADRB3 expression in plasma membranes from placental arteries obtained in normotensive (n = 3) and preeclamptic (n = 3) women. Expected size for ADRB3 is 68 kDa. Homogenate of CHO cells transfected with the human ADRB3 (ADRB3-CHO) was used as a positive control. B) Analysis of the expression of ADRB3 immunoreactive protein in placental arteries obtained from normotensive and preeclamptic women. The ADU represents the intensity of the bands evaluated by densitometry. Each bar represents the mean ± SEM from three different normotensive and three different preeclamptic women

Histological Evaluation of Placental Arteries

Histological analysis of placental arteries revealed no histological difference between arteries obtained from normotensive and preeclamptic women (data not shown). Furthermore, no alteration of endothelium after suspension in organ baths was observed.

DISCUSSION

The present results suggest, to our knowledge for the first time, that ADRB3 is present and functional in human placental arteries. Our results also confirm and extend the results published by Resch et al. [28], who described that ADRB2 agonists antagonized the electrical field stimulation (EFS)-induced contraction of the human placental arteries in a dose-dependent manner and that ADRB2 mRNAs were expressed in both placental veins and arteries. The functional and biochemical effects observed in the present study with SR 59119A are very likely to be mediated through an ADRB3 stimulation. Indeed, we have demonstrated previously the selectivity of this agonist for the ADRB3 in human colon and near-term myometrium [15–17, 21]. The pharmacological evidence for the presence of ADRB3 in human placental arteries also was supported by the use of ADRB antagonists. The SR 59119A-induced relaxation of human placental arteries was antagonized by the blockade of ADRB3 with 10 µM propranolol but not by the blockade of ADRB1 and ADRB2 with 0.1 µM propranolol [24]. The vasodilator effect of salbutamol on placental arteries is in general agreement with the findings of Dennedy et al. [23], who described a ritodrine-induced (ADRB2 agonist) relaxation in vitro on human umbilical arteries. Moreover, in Dennedy et al., it was found that BRL 37344, a preferential ADRB3 agonist, was responsible for concentration-dependent relaxation of human umbilical arteries. Some discrepancies are observed, however, between the study by Dennedy et al. and the present study, because we found that ADRB3 agonist-induced relaxation of human placental arteries is more pronounced than that induced by ADRB2 stimulation. This might be explained by the protocol differences (umbilical arteries for Dennedy et al. and placental arteries in the present study) and by the fact that BRL37344 was described, in human colon smooth muscle tissue, to be a weak agonist on ADRB3 compared with SR 59119A, the ADRB3 agonist used in the present study [15].

The present results show that SR 59119A and salbutamol were responsible for a significant increase in the cAMP level but failed to increase the cGMP level. The stimulation of cAMP production after exposure to SR 59119A was not modified by the presence of 0.1 µM propranolol, whereas salbutamol-induced cAMP production was inhibited after such pretreatment, providing confirmation of the ADRB3-mediated cAMP production. The relationship between cAMP production and smooth muscle relaxation has been well documented. Agents that increase the synthesis of cAMP (e.g., ADRB stimulants), as well as agents that inhibit cAMP degradation (e.g., phosphodiesterase inhibitors), all decrease smooth muscle contraction [26, 29]. Little is known about ADRB3 in vascular tissue, but the present results are in good agreement with those of Tamaoki et al. [30], who found that ADRB3 agonist-induced relaxation of dog pulmonary artery was mediated through a cAMP increase. An apparent contradiction can be observed, however, with the work of Dessy et al. [31], who found that ADRB3 is expressed in the endothelium of human coronary resistance arteries and mediates adrenergic vasodilatation through both NO and vessel hyperpolarization. The relaxant effect of SR 59119A on placental arteries observed in the present study is unlikely to be mediated through NO production, both because the CRC for SR 59119A was unaffected by the blockade of NO synthase by L-NNA and because we did not observe any SR 59119A-induced stimulation of cGMP production.

Pharmacological evidence for human placental artery ADRB3 was strengthened by detection of ADRB3 transcripts and protein. The ADRB3 was described to be located on endothelial cells in rat thoracic aorta [19, 32], but its exact localization in human placental arterial wall remains to be investigated.

We assessed the influence of pregnancy-induced hypertension on ADRB3 presence and responsiveness, a study that to our knowledge has not been done previously. Little is known about ADRB3 in preeclampsia, which is a condition associated with metabolic disorders, including insulin resistance [33] and changes of the metabolic syndrome [34], as well as with cardiovascular changes. A polymorphism of ADRB3 has been associated with the metabolic syndrome, including earlier onset of diabetes mellitus and obesity. This variant is a single substitution of arginine for tryptophan at position 64 [35]. Malina et al. [36], however, reported that the Trp64Arg polymorphism of ADRB3 does not predispose to preeclampsia.

Response of placental arteries to SR 59119A was attenuated in the preeclamptic compared to the normotensive group, whereas no difference in response to salbutamol and isoproterenol was observed. As shown in Western blot experiments, this impairment of the SR 59119A-induced cAMP production and relaxation occurs at the receptor level. Indeed, the density of ADRB3 proteins was decreased, whereas ADRB3 mRNA was expressed at the same level in arteries obtained from normotensive and preeclamptic pregnancies. This finding suggests that posttranscriptional downregulation of ADRB3 occurs. Such downregulation of ADRB already has been reported for the human ADRB2 [21], as has the absence of correlation between transcript and receptor expression (although not unanimously). Indeed, Lecrivain et al [37] described that treatment of late pregnant rats with high doses of isoproterenol (8 mg/kg twice daily) induced a selective decrease of ADRB2-binding sites, as assessed by 125I-cyanopindolol binding, and a rapid alteration of their transcript levels. In another study performed in human tissue, however, Engelhardt et al. [38], observed a 50% reduction in the number of ADRB2 in myometrial biopsyspecimens obtained from women treated with fenoterol before delivery compared with untreated women, whereas the mRNA concentrations of ADRB2, as determined by PCR, were unaffected by fenoterol treatment.

An impairment in the coupling of the receptor to adenylyl cyclase cannot formally be ruled out. Such modifications in the coupling mechanisms between ADRB2 and the catalytic component, implicated in the loss of ADRB/adenylyl cyclase stimulation, have been described in the myometrium at the end of pregnancy [39], or after prolonged stimulation with an ADRB2 agonist [37]. This has been related either to a decrease in the amount of functional Gs protein [39] or to an increased Gi activity [37].

Several studies have investigated the influence of preeclampsia on response of human placental arteries to different vasorelaxant agents, but the results reported have been contradictory. For example, Inayatulla et al. [40] concluded that the response of placental arteries to isoproterenol and to sodium nitroprusside, an NO donor, was not modified by preeclampsia, whereas Ong et al. [41] observed an alteration of vasorelaxation induced by sodium nitroprusside in placental arteries obtained from preeclamptic patients compared to normotensive women. Because sodium nitroprusside relaxes arteries independently of the endothelium, this finding is in agreement with ours. Indeed, we have shown that ADRB3 agonist-induced relaxation is endothelium independent and is altered in arteries obtained from preeclamptic women. Our findings on the fact that ADRB2 transcription and function were unaffected by preeclampsia seem to contradict the work by Aune et al. [10], who found that the number of functional ADRB2 was reduced in preeclampsia (390 ± 90 sites/cell in normal pregnancy vs. 80 ± 40 sites/cell in preeclampsia) because of a reduction in the total receptor number. This apparent discrepancy might be explained by the fact that the pharmacological characterization of ADRB2 was determined in isolated human mononuclear leukocytes instead of in placental arteries that are involved in the hypertension pathophysiology of preeclampsia. The absence of correlation between the ADRB2 density in leukocyte and pregnancy organ (e.g., uterus), is highlighted by the fact that Aune et al. [10] found a higher density of functional ADRB2 in leukocytes obtained at the end of uncomplicated pregnancies than in leukocytes obtained 6 wk after delivery. Such data suggest an increase of ADRB2 at the end of pregnancy. Breuiller et al. [42] and Rouget et al. [21], however, found that the myometrial ADRB2 density diminished at the end of pregnancy, reaching a low level at term. Because terms were not different for normal and preeclamptic pregnancies, the observed difference of ADRB3 expression and function is unlikely to be related to term. The absence of difference in terms at birth between the two groups of women is not a surprising finding, because Mostello et al. [43] reported that gestational age at delivery for preeclamptic pregnancies was more than 37 wk in close to 74% of the cases.

In conclusion, the present study provides compelling evidence for the presence of functional ADRB2 and ADRB3 in human placental arteries. Their stimulation induces a vasorelaxation in these vessels. In light of these findings and our recent data concerning ADRB3 resistance to desensitization [21] and overexpression in human term myometrium [17], we suggest that ADRB3 agonists may have considerable future pharmaceutical implications in the clinical management of pregnancy-related disorders (e.g., preterm labor) or other conditions (e.g., intrauterine growth restrictions) in which improvement in fetoplacental exchanges might be of potential interest. It can be argued that the therapeutic interest of ADRB3 agonists might, theoretically, be limited by the presence of this receptor in other vascular beds. Nevertheless, a recently published, placebo-controlled, randomized trial showed that a 28-day treatment with L-796568, an ADRB3 agonist, did not induce any significant cardiovascular changes [44]. Furthermore, it was described recently that the level of ADRB3 expression is higher in human placenta than in human aorta [45]. The consequences of the decreased number of human placental arteries ADRB3 immunoreactive proteins, associated with an impairment of ADRB3 function, observed in preeclampsia deserve further investigations.

ACKNOWLEDGMENTS

The authors thank the team of obstetrics and gynecology services at the University Hospital CHU du Bocage (Dijon, France) for its contribution. We also thank Dr. P. Marini (Sanofi-Aventis Research Center) for the kind gift of CHO cells transfected with the human ADRB3.

FOOTNOTES

1 Supported, in part, by the Fondation pour la Recherche Médicale and, in part, by the grant SAF2003–07206-C02 (Spain). Back

2 Correspondence: M. Bardou, Faculty of Medicine, LPPCE, 7 Bd Jeanne d'Arc, BP 87900, 21079 Dijon Cedex, France. FAX: 33 3 80 39 32 93; marc.bardou{at}u-bourgogne.fr Back

Received: 12 May 2005.

First decision: 30 May 2005.

Accepted: 21 September 2005.

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