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CARDIOVASCULAR
-Adrenergic Receptors in the Effect of the 
-Adrenergic Receptor Ligands, CGP 12177, Bupranolol, and SR 59230A, on the Contraction of Rat Intrapulmonary Artery
Institut National de la Santé et de la Recherche Médicale EMI-0356, Université Victor Segalen Bordeaux 2, Laboratoire de Pharmacologie de la Faculté de Pharmacie (V.L., F.P., B.M.) and Laboratoire de Physiologie Cellulaire Respiratoire (C.G., R.M.), Bordeaux, France; and Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Valencia, Spain (M.D.I.)
Received October 7, 2003; accepted December 19, 2003.
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Abstract |
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3- and/or atypical -adrenergic receptors (-AR) ligands] on the contractile function of rat intralobar pulmonary artery. Affinities of -AR ligands for 
1-adrenergic receptors (1-AR) were also evaluated using [3H]prazosin binding competition experiments performed in rat cortical membranes. In intralobar pulmonary artery, CGP 12177 did not modify the basal tone, but antagonized the contraction induced by the 1-AR agonist phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation, whereas in those precontracted with prostaglandin F2 (PGF2), it further enhanced contraction. CGP 12177 induced an increase in intracellular calcium concentration in pressurized arteries loaded with Fura PE-3 and precontracted with PGF2. In PGF2 precontracted arteries, phentolamine (an -AR antagonist) and phenoxybenzamine (an irreversible -AR antagonist) antagonized the contractile responses to PHE and CGP 12177. Both responses were also decreased by bupranolol and SR 59230A. Specific [3H]prazosin binding was displaced by CGP 12177, bupranolol, and SR 59230A with pKi values of 5.2, 5.7, and 6.6, respectively. In contrast, (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium (BRL 37344) and disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243) (nonaryloxypropanolamines 3-AR agonists) displayed very low affinity for [3H]prazosin binding sites (pKi values below 4). These data suggest that CGP 12177 exhibits partial agonist properties for 1-AR in rat pulmonary artery. They also show that bupranolol and SR 59230A exert an 1-AR antagonist effect. As a consequence, these aryloxypropanolamine compounds should be used with caution when investigating the role of 3- and atypical -AR in the regulation of vascular tone.
-adrenergic receptors (-AR) studies. CGP 12177 was initially described as a high affinity antagonist of 1- and 2-AR (Staehelin et al., 1983
; Nanoff et al., 1987). Later, it was shown to interact, with a lower affinity, with the 3-AR and to exhibit a partial agonist activity on rodent and human 3-AR (Feve et al., 1991; Granneman et al., 1991; Blin et al., 1993). However, at least two responses induced by CGP 12177, which were initially thought to be mediated by the 3-AR (namely, the cardiostimulant response and some lipolytic effects) were still observed in 3-AR knockout mice (Kaumann et al., 1998; Preitner et al., 1998). The existence of a novel -AR subtype, initially named "putative 4-AR subtype", was then postulated to account for these 3- AR-independent effects of CGP 12177. The term of atypical -AR has emerged to define receptors that display pharmacological properties different from those of 1-, 2-, and 3-AR (Molenaar, 2003). These atypical -AR are proposed as distinct states of the 1-AR, in particular a low affinity state (l.a.s. 1-AR) (Konkar et al., 2000a,b; Lowe et al., 2002). Therefore, CGP 12177 is referred to as a nonconventional partial agonist, with agonist properties on 3- and atypical -AR, but antagonist on 1- and 2-AR. To further distinguish 3- from atypical -AR-mediated responses, other compounds are commonly used, such as the aryloxypropanolamine derivatives bupranolol (a nonselective -AR antagonist), SR 59230A (a selective 3-AR antagonist), and the phenylethanolamine compounds BRL 37344 and CL 316243 (selective 3-AR agonists) (Granneman, 2001).
A role of -AR, distinct from 1- and 2-AR, in modulating vascular tone was initially supported by in vivo studies in dogs describing a peripheral vasodilatation in response to administration of BRL 37344, CL 316243, and CGP 12177 (Berlan et al., 1994; Shen et al., 1996). Subsequent studies were conducted in vitro in isolated arteries. 3-AR agonists were shown to relax the rat carotid artery (Oriowo, 1994; MacDonald et al., 1999). In rat aorta precontracted with phenylephrine (PHE), 3-AR agonists induced an endothelium- and NO-dependent vasorelaxant effect (Trochu et al., 1999). The expression of mRNA and protein of the 3-AR was recently demonstrated in rat aortic endothelial cells (Rautureau et al., 2002). The presence of a functional -AR, presenting more pharmacological similarities with the atypical -AR than with the 3-AR, was also reported in rat aorta (Shafiei and Mahmoudian, 1999; Brawley et al., 2000). However, a recent study provided no evidence for the presence of functional 3-AR or l.a.s. 1-AR in rat aorta (Brahmadevara et al., 2003). Indeed, the authors observed that CGP 12177 and BRL 37344 elicited relaxation in PHE-precontracted aortic rings, but failed to do so in those precontracted with prostaglandin F2 (PGF2). This study raises the question of the choice of the preconstrictor agent for investigation of -AR-mediated vasorelaxation and suggests a possible interference of some -AR ligands with the -AR signaling pathway (Brahmadevara et al., 2003). The influence of the vasoconstrictor agent on the responses induced by 3- and atypical -AR agonists was also noticed in rat mesenteric artery (Kozlowska et al., 2003). According to these data, it appears that the presence of functional 3- and atypical -AR in arteries are still under debate. Moreover, most of these studies were performed in arteries of systemic circulation, and relatively little information is available in pulmonary circulation. In canine large pulmonary artery, the existence of a vasorelaxant 3-AR was proposed (Tamaoki et al., 1998). In rat isolated perfused lung, it was shown that some selective 3-AR agonists (phenylethanolaminotetraline compounds) opposed the hypoxic vasoconstriction (Dumas et al., 1998). However, the receptor involved in this effect did not fully match the 3-AR subtype.
The aim of the present study was therefore to evaluate the effect of CGP 12177 and the other aryloxypropanolamine -AR ligands, bupranolol and SR 59230A, on the contraction of isolated rat intralobar pulmonary artery and to investigate a possible interaction of these -AR ligands with the 1-AR. For this purpose, the functional responses induced by CGP 12177 were characterized in the absence and presence of - or -AR antagonists, and compared with those of PHE, the reference 1-AR agonist. The affinity of CGP 12177 and several 3- and atypical -AR ligands toward the 1-AR was also determined using [3H]prazosin binding competition experiments. Preliminary accounts of this work have previously been published in abstract form (Leblais et al., 2003).
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Materials and Methods |
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). To determine this resting tension, a passive length-tension relationship was generated for each intralobar pulmonary artery. In the case of extralobar pulmonary arteries, vessels were progressively stretched to a resting tension of 3.7 mN. After 60 min of equilibration period under resting tone, viability of arteries was evaluated using PSS containing 80 mM KCl (equimolar substitution with NaCl). Intralobar preparations developing a wall tension below 1 mN/mm were discarded.
After washouts, cumulative concentration-response curves were performed with either CGP 12177 (10 nM–100 µM) or the 1-AR agonist PHE (1 nM–100 µM) under different experimental conditions. In a first procedure, CGP 12177 or PHE was applied on the resting tone. In experiments evaluating the effect of CGP 12177 on the response induced by PHE, two consecutive concentration-response curves to PHE were performed. The second curve was conducted in the absence or presence of CGP 12177 (100 µM) and added 15 min before PHE. In a second procedure, the concentration-response curves to CGP 12177 or PHE were performed in arteries precontracted with PGF2 (3 or 30 µM, concentrations corresponding approximately to EC15 and EC80, respectively). When indicated, preparations were incubated with the -AR antagonist phentolamine (1 µM), bupranolol (5 µM), or SR 59230A (1 or 3 µM) for 25 min before carrying out the concentration-response curve to CGP 12177 or PHE. To irreversibly inactivate -AR, arteries were pretreated by the alkylating agent phenoxybenzamine (PBZ) at 1 µM for 15 min. After PBZ pretreatment, arteries were washed with PSS, and experiments were performed as described above. In a third procedure, the CGP 12177 concentration-response curve was conducted in arteries precontracted with PHE (30 nM or 3 µM, concentrations corresponding approximately to EC20 and EC80, respectively). At the end of each procedure, the presence of a functional endothelium was evaluated by the relaxant effect in response to application of acetylcholine (10 µM).
Measurements of Intracellular Calcium and External Arterial Diameter. Intralobar pulmonary arteries were removed from male Wistar rats, as described above. Segments of 1-mm length were cannulated at both ends with glass micropipettes, secured with 10-0 nylon monofilament suture, and placed in a microvasculature chamber (Living Systems, Burlington, VT). Using a peristaltic pump and a pressure sensor, vessels were held at a constant transmural pressure of 15 mm Hg. The chamber was superfused at a rate of 2 ml/min with Krebs-Hepes solution [containing 118.4 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4 mM NaHCO3, 10 mM Hepes, and 6 mM glucose; pH = 7.4 with NaOH] gassed with air, and maintained at 37°C.
Arteries were loaded with Fura PE-3 AM (an analog of Fura-2) by incubation in the microvascular chamber with 1 µM Fura PE-3 AM in Krebs-Hepes solution for 90 min at 37°C. After washout with Krebs-Hepes solution, the chamber was placed on the stage of an inverted epifluorescence microscope (IX70; Olympus, Rungis, France) equipped with a x10, UplanApo 0.4 W water immersion objective (Olympus). The source of excitation light was a xenon arc lamp (175 W). Fura PE-3 AM was alternately excited at two wavelengths (345 and 380 nm) selected by a monochromator (Life Science Resources, Cambridge, UK). Digital images were sampled at 12-bit resolution by a fast scan cooled charge-coupled device camera (Cool-SNAP fx monochrome; Photometrics, Paris, France). Ratios of the 345- to 380-nm images (345/380) were produced every 20 s. All the imaging was controlled by Universal Imaging software including metafluor and metamorph (Universal Imaging Corporation, Downingtown, PA). Regions of interest were drawn on the vascular wall to quantify the ratio 345:380, which is an index of the intracellular calcium concentration. To determine the external diameter of the vessel, three transversal lines were drawn on the ratio image. The mean number of fluorescent pixels crossing those lines was calculated by metamorph software and used for external diameter recording. Arteries were first perfused in the presence of PGF2 (3 µM) during 10 min and then with increasing concentrations of CGP 12177 (1–100 µM) still in the presence of PGF2.
[3H]Prazosin Binding Assay. After decapitation, the brain was rapidly removed from female Wistar rats (180–200 g, 12 weeks old, obtained from Harlan Interfauna Ibérica, Barcelona, Spain). Cerebral cortex membranes were prepared as previously described (Madrero et al., 1996). Protein concentration was determined according to the method of Bradford (1976) using globulin as standard. Assays of competition of [3H]prazosin binding were performed in aliquots of diluted membranes (300 µg of proteins per tube) incubated in 50 mM Tris buffer (pH 7.5) with 0.2 nM [3H]prazosin (specific activity 84 Ci/mmol) in the absence or presence of various concentrations of competitors. Incubations (total volume of 1 ml) were performed for 45 min at 25°C under continuous shaking. Bound [3H]prazosin was separated from the free [3H]prazosin by filtration using a Brandel cell harvester (M24R) through glass fiber filters (No. 30; Schleicher and Schuell, Keene, NH) presoaked with 0.3% polyethylenimine, followed by three washes with ice-cold 50 mM Tris buffer. Filter-bound radioactivity was determined by liquid scintillation counting. Nonspecific binding was defined as [3H]prazosin binding in the presence of phentolamine (10 µM). Each assay was performed in duplicate.
Drugs. Acetylcholine chloride, BRL 37344 sodium salt, (±)-CGP 12177 hydrochloride, CL 316243, PGF2 Tris salt, phenoxybenzamine hydrochloride, phentolamine hydrochloride, (-)-phenylephrine hydrochloride, SR 59230A oxalate salt, and Fura PE-3 AM were supplied by Sigma Chemical Co. (St Quentin-Fallavier, France). Bupranolol was obtained from Schwarz Pharma (Monheim, Germany). 7-Methoxy-[3H]prazosin was obtained from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). All drugs were prepared as stock solution in distilled water, with the exception of SR 59230A and Fura PE-3 AM, which were dissolved in dimethyl sulfoxide (Sigma Chemical Co.) so that the final concentration of the solvent in contact with arteries was less than 0.05% for SR 59230A and less than 0.1% for Fura PE-3 AM.
Data Analysis. Isometric tension is expressed either as percentage of the initial tone (induced by PGF2 or PHE) or percentage of the response to 80 mM KCl, as appropriate. To determine the potency of agonist, the EC50 value (concentration that produces 50% of the maximum response) was estimated in each individual concentration-response curve using the Boltzman equation fit and converted to negative logarithm value (pD2). EC15, EC20, and EC80 were defined as the concentration of agonist that produces 15, 20, and 80% of the maximum response, respectively. In the case of the CGP 12177 concentration-response curve, the EC50 value could not be determined because the maximum response was not reached at the highest concentration used. To quantify the potency of antagonists, estimated pKB values were calculated according to the equation pKB = log(r - 1) - log[B], where r is the ratio of the mean EC50 values in the presence and absence of antagonist, and B the antagonist concentration.
Ligand competition curves were analyzed by nonlinear regression (one-site model) with Graph-Pad PRISM. pKi values were determined according to the Cheng and Prusoff equation (Cheng and Prusoff, 1973).
Data are given as means ± S.E.M. Data were compared using a Student's t test or a two-way analysis of variance (ANOVA) as appropriate. Differences were considered statistically significant when P < 0.05.
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or 30 nM PHE (corresponding approximately to the EC15 and EC20, respectively). The tension developed in response to these agents was not significantly different [tension: 14.6 ± 1.2% of response to 80 mM KCl (n = 14) with PGF2 compared with 17.5 ± 4.9% (n = 4) with PHE]. In arteries precontracted with 3 µM PGF2, addition of increasing concentrations of CGP 12177 further enhanced tension in a concentration-dependent manner (Fig. 1, A and C). In contrast, in arteries precontracted with 30 nM PHE, CGP 12177 induced a concentration-dependent relaxation (Fig. 1, B and C). Concentrations of CGP 12177 producing either contraction or relaxation were within the same range. A similar pattern of responses to CGP 12177 was still noticed when the concentration of precontractile agent was increased, namely 30 µM PGF2 or 3 µM PHE (inducing a comparable level of contraction and corresponding to the EC80 value of each agonist). However, the amplitude of the responses were smaller: for 100 µM CGP 12177, tension reached 133 ± 5% of PGF2-induced tone (n = 6) and 80 ± 5% of PHE-induced tone (n = 5) (data not shown). The differential effect of CGP 12177 depending on the precontractile agent was not only observed in small intralobar pulmonary arteries but also in large extralobar pulmonary arteries (data not shown). In addition, CGP 12177 elicited similar qualitative and quantitative responses in intralobar pulmonary arteries removed from male or female rats. In arteries removed from female rats, the tension reached after addition of 100 µM CGP 12177 was 305 ± 13% of PGF2 (3 µM)-induced tone (n = 3) and 74 ± 8% of PHE (30 nM)-induced tone (n = 3). These observations suggested that CGP 12177 may interact with 1-AR in rat pulmonary arteries. In subsequent experiments, the contractile effect of CGP 12177 was systematically compared with the one of PHE, the reference 1-AR agonist.
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Influence of Tone on the Contractile Effect of CGP 12177 and PHE in Rat Intralobar Pulmonary Artery. When applied on the basal tone, CGP 12177 exerted only minor effect in rat intralobar pulmonary arteries (Fig. 2A). By contrast, PHE caused a concentration-dependent contraction with a pD2 value of 6.3 ± 0.1 (n = 23, Fig. 2B). Precontraction with PGF2 (3 µM) uncovered the contractile effect of CGP 12177 and markedly enhanced the PHE-induced contraction (Fig. 2, A and B). In PGF2-precontracted arteries, the pD2 value of PHE was significantly increased (pD2 = 7.7 ± 0.1, n = 6, P < 0.001 in comparison to pD2 value of PHE on the basal tone), but its maximal response was not modified.
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Effect of CGP 12177 on Intracellular Calcium Concentration in Rat Intralobar Pulmonary Artery. In pressurized intralobar pulmonary arteries loaded with Fura PE-3, PGF2 (3 µM) induced an increase in intracellular calcium concentration (Fig. 3A) and a vasoconstriction (Fig. 3B). Addition of CGP 12177 (1–100 µM) on PGF2-precontracted arteries induced a further increase in the intracellular calcium concentration (Fig. 3A) and contraction (Fig. 3B).
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Effect of CGP 12177 on the Contractile Response of PHE in Rat Intralobar Pulmonary Artery. The influence of CGP 12177 on the PHE-induced contraction was analyzed in non-precontracted intralobar pulmonary arteries. A 15-min pretreatment with 100 µM CGP 12177 did not significantly affect the basal tone [4.7 ± 1.8% of response to 80 mM KCl (n = 8) compared with 2.5 ± 1.7% in the absence of CGP 12177 (n = 7)]. In the presence of 100 µM CGP 12177, the concentration-response curve to PHE was significantly shifted to the right [pD2 = 7.1 ± 0.1 (n = 7) in the absence of CGP 12177, compared with 5.6 ± 0.1 (n = 8) in the presence of CGP 12177, P < 0.01] without any changes in the maximal response (Fig. 4). The pKB value of CGP 12177 was estimated as 5.3.
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Effect of -AR Antagonists on the Contractile Effect of CGP 12177 and PHE in PGF2-Precontracted Rat Intralobar Pulmonary Artery. The potential role of -AR in CGP 12177-induced contractile response was further investigated using -AR antagonists. As expected, phentolamine (1 µM), a competitive -AR antagonist, did not modify the tension induced by 3 µM PGF2 [tension: 18.0 ± 2.3% of response to 80 mM KCl (n = 12) in the presence of phentolamine, compared with 14.6 ± 1.2% (n = 14) in the absence of phentolamine] but significantly shifted to the right the concentration-response curve to PHE (Fig. 5B) [pD2 = 5.8 ± 0.01 (n = 6) in the presence of 1 µM phentolamine, compared with 7.7 ± 0.1 (n = 6) in the absence of phentolamine, P < 0.001]. The pKB value of phentolamine was estimated as 7.9. Phentolamine also inhibited CGP 12177-induced contractile response in PGF2-precontracted arteries (Fig. 5A). The effect of another -AR antagonist, the alkylating agent PBZ that irreversibly inactivates -AR, was also studied. A 15-min pretreatment with 1 µM PBZ had no significant effect on the PGF2-induced contraction [tension: 19.3 ± 3.0% of response to 80 mM KCl (n = 8) after PBZ, compared with 14.6 ± 1.2% (n = 14) in the absence of PBZ]. PBZ pretreatment abolished the PHE-induced vasoconstriction in PGF2-precontracted arteries (Fig. 6B) and markedly reduced the CGP 12177-induced contractile response (Fig. 6A).
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Effect of Atypical -AR Antagonists on the Contractile Effect of CGP 12177 and PHE in PGF2-Precontracted Rat Intralobar Pulmonary Artery. Bupranolol and SR 59230A are two aryloxypropanolamines commonly used as atypical -AR antagonists. At the concentration of 5 µM, bupranolol (a nonselective -AR antagonist) did not change the PGF2-induced tone [tension: 18.0 ± 2.6% of response to 80 mM KCl (n = 12) in the presence of bupranolol, compared with 14.6 ± 1.2% (n = 14) in the absence of bupranolol] but significantly decreased the CGP 12177-induced contraction (Fig. 7A). Furthermore, bupranolol caused a rightward shift of the concentration-response curve to PHE without modifying the maximal response (Fig. 7B). The pD2 value of PHE decreased from 7.7 ± 0.1 (n = 6) in the absence of bupranolol to 6.9 ± 0.2 in the presence of bupranolol (n = 6, P < 0.01). The estimated pKB value of bupranolol was 6.0.
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In the presence of 3 µM SR 59230A (a drug commonly described as a selective 3-AR antagonist), the PGF2-induced tone was significantly increased [tension: 20.4 ± 2.7% of response to 80 mM KCl (n = 8) in the presence of 3 µM SR 59230A, compared with 14.6 ± 1.2% (n = 14) in the absence of SR 59230A, P < 0.05] and the contractions induced by CGP 12177 and PHE were markedly depressed (Fig. 8, A and B). When the concentration of SR 59230A was reduced to 1 µM, the PGF2-induced tone was not significantly altered [tension: 13.5 ± 1.1% of response to 80 mM KCl (n = 13) in the presence of 1 µM SR 59230A, compared with 14.6 ± 1.2% (n = 14) in the absence of SR 59230A] but the contractile response to CGP 12177 was still significantly inhibited, albeit to a lesser extent than with 3 µM SR 59230A (Fig. 8A). SR 59230A (1 µM) also caused a significant rightward shift of the PHE concentration-response curve (Fig. 8B). The pD2 value of PHE diminished from 7.7 ± 0.1 (n = 6) in the absence of SR 59230A to 6.4 ± 0.2 in the presence of SR 59230A (n = 6, P < 0.01), whereas the maximum contraction was not changed. The estimated pKB value of SR 59230A was 7.3.
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Affinity of Different AR Ligands for [3H]Prazosin Binding Sites. The affinities of some - and -AR ligands toward 1-AR were determined by [3H]prazosin-binding competition assays. As expected, phentolamine, the reference -AR antagonist, completely inhibited the specific binding of [3H]prazosin (Fig. 9 and Table 1). SR 59230A, bupranolol, and CGP 12177 also fully and concentration-dependently displaced [3H]prazosin-specific binding, with pKi values ranging from 5.2 to 6.6 (Table 1). In contrast to these aryloxypropanolamine compounds, the phenylethanolamine derivatives BRL 37344 and CL 316243 competed with [3H]prazosin binding sites at concentrations 
100 µM (Fig. 9 and Table 1).
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Discussion |
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1-AR. CGP 12177 elicited a relaxation in PHE-precontracted arteries, had negligible effect when applied on basal tone, but further enhanced contraction and intracellular calcium concentration in PGF2-precontracted preparations. CGP 12177-induced contraction was inhibited by phentolamine and PBZ (-AR antagonists) and also by bupranolol and SR 59230A. Moreover, CGP 12177, bupranolol, and SR 59230A antagonized PHE-induced contraction. Finally, it is shown that CGP 12177, bupranolol, and SR 59230A exhibited affinity for [3H]prazosin binding sites.
In this study, CGP 12177 induced relaxation for concentrations 1 µM in PHE-precontracted arteries. This is consistent with the potency of CGP 12177 in other rat arteries (Trochu et al., 1999; Brahmadevara et al., 2003; Kozlowska et al., 2003). Recent reports highlight the influence of the precontractile agonist on the amplitude of the relaxant effect of some -AR agonists. For instance, CGP 12177 produced relaxation in PHE-precontracted rat aorta, but failed to do so in a PGF2-precontracted one (Brahmadevara et al., 2003). Similarly, the relaxation evoked by cyanopindolol (another aryloxypropanolamine, nonconventional partial agonist) in rat mesenteric artery was strongly reduced in vessels constricted with PGF2, compared with those precontracted with PHE (Kozlowska et al., 2003). In the present study, the effect of CGP 12177 was also evaluated in arteries precontracted with PGF2 instead of PHE. Surprisingly, in these arteries, CGP 12177 exerted no relaxant effect, but rather induced contraction. These two opposite effects (contraction or relaxation) were observed within the same range of concentrations, suggesting a common underlying mechanism. To our knowledge, it is the first time that a contractile effect of CGP 12177 is reported. The use of relatively low concentrations of PGF2 (producing only 15% of its maximum response) might explain why contractile effects of CGP 12177 could be unmasked here. Accordingly, increasing precontraction level resulted in a marked reduction in the amplitude of CGP 12177-induced contraction.
Interference of CGP 12177 with -AR or -AR-mediated intracellular signaling pathway has been postulated from experiments showing that CGP 12177 produced relaxation in PHE-precontracted rat aorta, but not in those precontracted with PGF2 (Brahmadevara et al., 2003). The present study provides evidence for an interaction of CGP 12177 at the level of 1-AR. Our experimental data support the idea that 1-AR agonist properties contributed to the contractile effect of CGP 12177 in PGF2-precontracted arteries. The increase in intracellular calcium concentration evoked by CGP 12177 is consistent with 1-AR-mediated response. Moreover, CGP 12177-induced contraction was diminished by phentolamine and PBZ. Since the latter compounds are nonselective -AR antagonists, a potential contribution of 2-AR in the effect of CGP 12177 cannot be excluded. Both phentolamine and PBZ also inhibited the effect of the selective 1-AR agonist PHE. With phentolamine, an apparent competitive antagonism was obtained and its estimated pKB value (7.9) is consistent with the affinity for 1-AR determined in the present study (pKi value 7.8) and in literature (Yan et al., 2001). Even though functional and binding experiments were performed in different rat tissues (intralobar pulmonary artery and cerebral cortex) and in animals from different sexes, the affinity of phentolamine determined by these two approaches was very similar. With the irreversible -AR antagonist PBZ, PHE-induced contraction was abolished, whereas CGP 12177-induced contraction was partially inhibited. If the effect of CGP 12177 was exclusively due to the activation of -AR, it would be expected to be also totally abolished by PBZ. Thus, CGP 12177-induced contraction can be attributed to -AR activation and to an -AR-independent component. The nature of the latter deserves further investigation.
The lack of effect of CGP 12177 on basal tone (i.e., in the absence of PGF2) seems a priori not consistent with 1-AR agonist properties of this compound. However, in conditions that unmasked the contractile properties of CGP 12177, PGF2 increased the sensitivity to PHE. Different mechanisms may underlie PGF2-induced potentiation of 1-AR-mediated response. It has been reported that the vasoconstriction induced by stimulation of some receptors can be enhanced, or even uncovered, by increasing tone through Gq protein-coupled receptor pathway (Choppin and O'Connor, 1995; Movahedi and Purdy, 1997). Several studies have shown that the vasoconstriction induced by -AR stimulation can be potentiated by agonists of other G protein-coupled receptors (Fabi et al., 1998; Bhattacharya and Roberts, 2003). Different mechanisms are proposed to explain this kind of potentiation, like tyrosine kinase/protein kinase C-dependent pathway (Henrion and Laher, 1994), a RhoA/Rho kinase pathway (Boer et al., 2002), or the extracellular signal-regulated kinase/mitogen-activated protein kinase cascade (Bhattacharya and Roberts, 2003). The implication of these pathways in enhanced sensitivity to PHE in PGF2- precontracted pulmonary arteries remains to be investigated. This phenomenon could have relevant pathological implications, especially in pulmonary hypertension, which is characterized by an increase in vascular tone (Strange et al., 2002). Whatever the mechanisms of potentiation, the disclosure of the contractile properties of CGP 12177 by PGF2 could be related to a low intrinsic efficacy of CGP 12177 on 1-AR. Indeed, one would expect that conditions producing potentiation of the effect of a full agonist would uncover the response to a partial agonist. As a partial agonist behaves as an antagonist in the presence of a full agonist (Kenakin, 1993), we also investigated the antagonist properties of CGP 12177 for 1-AR. In conditions in which it had only a minor effect per se on contraction (i.e., in the absence of precontractile agent), CGP 12177 antagonized PHE-induced contraction in an apparent competitive manner. These data support the idea that in rat intralobar pulmonary artery, CGP 12177 is a partial agonist of 1-AR. Competition for [3H]prazosin binding sites clearly demonstrated the 1-AR ligand properties of CGP 12177. It is noteworthy that the affinity of CGP 12177 determined by binding or functional assays were very similar (pKi and pKB values of 5.2 and 5.3, respectively). This further corroborates the implication of 1-AR in the effect of CGP 12177 in rat pulmonary artery.
In this study, other aryloxypropanolamines were evaluated as potential -AR ligands. Recently, it has been reported that bupranolol (a nonselective -AR antagonist) and SR 59230A (a selective 3-AR antagonist) elicited relaxation in PHE-precontracted rat aorta, but not in a PGF2-precontracted one (Brahmadevara et al., 2003). This suggests that, like CGP 12177, bupranolol and SR 59230A interfere with the 1-AR signaling pathway (Brahmadevara et al., 2003). The present study shows that bupranolol and SR 59230A antagonized PHE-induced contraction in intralobar pulmonary artery (pKB values of 6.0 and 7.3, respectively) and displaced specific [3H]prazosin binding in cortex cerebral membranes (pKi values of 5.7 and 6.6, respectively). These data support the competitive antagonist properties of bupranolol and SR 59230A at 1-AR. At concentrations >1 µM, SR 59230A markedly depressed the maximal response to PHE. Thus, additional mechanisms to -AR competitive antagonism are probably involved in the effect of SR 59230A observed here, especially at concentrations >1 µM. In some smooth muscles, SR 59230A has been reported to exert relaxant effects through agonist properties on 3-AR and/or atypical -AR (Horinouchi and Koike, 2001). However, it is unlikely that such 3-AR and/or atypical -AR agonists properties accounted for the antagonist effect of SR 59230A seen here since SR 59230A did not antagonize, but enhanced PGF2- induced contraction. It should be noted that in conditions producing inhibition of PHE-induced contraction, bupranolol and SR 59230A also diminished CGP 12177-induced contraction. This further supports the role of 1-AR in the effect of CGP 12177 in pulmonary artery. The relative contribution of 1-AR-dependent and -independent mechanisms to the effect of these aryloxypropanolamines in the present and other vascular models deserves further investigation.
Competition experiments presented here show that, in contrast to CGP 12177, bupranolol, and SR 59230A, BRL 37344 and CL 316243 displayed very low affinity for 1-AR. Interestingly, BRL 37344 and CL 316243 exhibited weak potency and efficacy in relaxing PHE-precontracted rat mesenteric arteries (Kozlowska et al., 2003). This might be related to the weak displacement of PHE from vascular 1-AR by CL 316243 and BRL 37344, owing to the low binding affinity of these two latter agents for 1-AR. CL 316243 and BRL 37344 are phenylethanolamine derivatives, whereas CGP 12177, bupranolol, and SR 59230A are aryloxypropanolamine (Nisoli et al., 1996; Strosberg, 1997). Aryloxypropanolamines likely share structural features responsible for interaction with -AR. A detailed structure-activity relationship would be necessary to clarify the molecular requirements for concomitantly binding to - and -AR. Nevertheless, phenylethanolamine derivatives appear more suitable tools than aryloxypropanolamines to investigate the role of 3-AR in the vasculature.
In conclusion, this study gives more insight into the pharmacological properties of several -AR ligands. It shows the affinity of CGP 12177, bupranolol, and SR 59230A for 1-AR. It also indicates that bupranolol and SR 59230A display antagonist effect toward 1-AR in rat pulmonary artery. Finally, it demonstrates that in this artery, CGP 12177 exhibits partial 1-AR agonist activity. Aryloxypropanolamine compounds should be used with caution to investigate the role of 3- and atypical -AR in the regulation of vascular contraction.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CGP 12177, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; AR, adrenergic receptor; l.a.s., low affinity state; SR 59230A, 3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate; BRL 37344, (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium; PBZ, phenoxybenzamine; CL 316243, disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate; PGF2, prostaglandin F2; PHE, phenylephrine; PSS, physiological salt solution; pD2, negative logarithm of E50 value; PE-3, pentakisester-3.
Address correspondence to: Dr. Véronique Leblais, Laboratoire de Pharmacologie de la Faculté de Pharmacie, INSERM EMI-0356, Université Victor Segalen Bordeaux 2—Casier 83, 146 rue Léo Saignat, 33076 Bordeaux cedex, France. E-mail: veronique.leblais{at}phcodyn.u-bordeaux2.fr
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