Role of -Adrenergic Receptors in the Effect of the - Adrenergic Receptor Ligands, CGP 12177, Bupranolol, and SR 59230A, on the Contraction of Rat Intrapulmonary Artery

This study investigates the effect of the aryloxypropanolamines 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro2H-benzimidazol-2-one (CGP 12177), bupranolol, and 3-(2ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2propanol oxalate (SR 59230A) [commonly used as 3and/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 [H]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 [H]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 [H]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 3and atypical -AR in the regulation of vascular tone. The aryloxypropanolamine 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177) is extensively used in the field of -adrenergic receptors ( -AR) studies. CGP 12177 was initially described as a high affinity antagonist of 1and 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 3AR-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. The authors thank the “Association des Enseignants de Pharmacologie des Facultés de Pharmacie” for partially supporting the collaboration between Valencia and Bordeaux Faculties. This work was also partially supported by a research grant from the “Generalitat Valenciana” (GV01-292). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.061192. 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. 0022-3565/04/3091-137–145$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 309, No. 1 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 61192/1136360 JPET 309:137–145, 2004 Printed in U.S.A. 137 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from 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 3and atypical -AR, but antagonist on 1and 2-AR. To further distinguish 3from 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 1and 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 endotheliumand 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 -ARmediated 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 3and 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 3and 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 3and atypical -AR ligands toward the 1-AR was also determined using [H]prazosin binding competition experiments. Preliminary accounts of this work have previously been published in abstract form (Leblais et al., 2003). Materials and Methods Measurements of Isometric Tension. Male Wistar rats (450– 600 g, 11–18 weeks old, obtained from Elevage Janvier, Le Genest Saint Isle, France) and, for some experiments, female Wistar rats (290–310 g, 15 weeks old, obtained from Elevage Janvier) were killed by CO2 inhalation. After exsanguination, the heart and lungs were excised and placed in a physiological salt solution (PSS) containing 119 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl2, 1.17 mM MgSO4, 1.18 mM KH2PO4, 25 mM NaHCO3, and 5.5 mM glucose. Intralobar pulmonary arteries (2nd–3rd order branch, internal diameter: 450– 850 m) and, in some cases, extralobar pulmonary arteries (left and right main branches) were dissected free of connective tissue. Segments of 1.6to 2-mm length were mounted in a Mulvany myograph (Multi Myograph system, model 610M; J. P. Trading, Aarhus, Denmark), bathed in PSS maintained at 37°C, and gassed with a mixture of 95% O2/5% CO2 (pH 7.4). The optimal resting tension for rat intralobar pulmonary arteries corresponds to an equivalent transmural pressure of 30 mm Hg (Leach et al., 1992). 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 M) or the 1-AR agonist PHE (1 nM–100 M) 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 M) 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 M, concentrations corresponding approximately to EC15 and EC80, respectively). When indicated, preparations were incubated with the -AR antagonist phentolamine (1 M), bupranolol (5 M), or SR 59230A (1 or 3 M) 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 M 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 M, 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 M). 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 M Fura PE-3 AM 138 Leblais et al. at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from 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 10, 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 (CoolSNAP fx monochrome; Photometrics, Paris, France). Ratios of the 345to 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 M) during 10 min and then with increasing concentrations of CGP 12177 (1–100 M) still in the presence of PGF2 . [H]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 [H]prazosin binding were performed in aliquots of diluted membranes (300 g of proteins per tube) incubated in 50 mM Tris buffer (pH 7.5) with 0.2 nM [H]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 [H]prazosin was separated from the free [H]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. Filterbound radioactivity was determined by liquid scintillation counting. Nonspecific binding was defined as [H]prazosin binding in the presence of phentolamine (10 M). Each assay was performed in dupli-