JPET #61192
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Role of α-adrenergic receptors in the effect of the ß-adrenergic
receptors ligands, CGP 12177, bupranolol and SR 59230A, on the
contraction of rat intrapulmonary artery.
Véronique LEBLAIS, Fabrice POURAGEAUD, M. Dolores IVORRA,
Christelle GUIBERT, Roger MARTHAN, and Bernard MULLER
INSERM EMI-0356, Université Victor Segalen Bordeaux 2, Laboratoire de Pharmacologie de
la Faculté de Pharmacie (V.L., F.P., B.M.) & Laboratoire de Physiologie Cellulaire
Respiratoire (C.G., R.M.), Bordeaux, France
Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Valencia, Spain
(M.D.I.)
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Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title:
Role of α-AR in aryloxypropanolamine effects.
Corresponding author:
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
Phone number: 33 5 57 57 12 01
Fax number: 33 5 57 57 12 01
e-mail: [email protected]
Number of - text pages: 26
- table: 1
- figures: 9
- references: 40
Number of words in - abstract: 246
- introduction: 734
- discussion: 1500
Abbreviations :
α-AR, α-adrenergic receptor; β-AR, β-adrenergic receptor; l.a.s.β1-AR, low affinity state of
the β1-AR; PBZ, phenoxybenzamine; PGF2α, prostaglandin F2α; PHE, phenylephrine; PSS,
physiological salt solution
Recommended section assignment:
Cardiovascular
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Abstract
This study investigates the effect of the aryloxypropanolamines CGP 12177, bupranolol and
SR 59230A (commonly used as β3- and/or atypical β-adrenergic receptors (β-AR) ligands) on
the contractile function of rat intralobar pulmonary artery. Affinities of β-AR ligands for α1-
adrenergic receptors (α1-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 α1-AR agonist
phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation
whereas in those precontracted with prostaglandin F2α (PGF2α), it further enhanced
contraction. CGP 12177 induced an increase in intracellular calcium concentration in
pressurized arteries loaded with Fura-PE3 and precontracted with PGF2α. In PGF2α-
precontracted arteries, phentolamine (an α-AR antagonist) and phenoxybenzamine (an
irreversible α-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, BRL 37344 and CL 316243 (non-aryloxypropanolamines
β3-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 α1-AR in rat
pulmonary artery. They also show that bupranolol and SR 59230A exert α1-AR antagonist
effect. As a consequence, these aryloxypropanolamine compounds should be used with
caution when investigating the role of β3- and atypical β-AR in the regulation of vascular
tone.
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The aryloxypropanolamine CGP 12177 is extensively used in the field of β-adrenergic
receptors (β-AR) studies. CGP 12177 was initially described as a high affinity antagonist of
both β1- and β2-AR (Staehelin et al., 1983; Nanoff et al., 1987). Later, it was shown to
interact, with a lower affinity, with the β3-AR and to exhibit a partial agonist activity on
rodent and human β3-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 β3-AR (namely, the cardiostimulant response and some lipolytic effects) were
still observed in β3-AR knockout mice (Kaumann et al., 1998; Preitner et al., 1998). The
existence of a novel β-AR subtype, initially named “putative β4-AR subtype”, was then
postulated to account for these β3-AR-independent effects of CGP 12177. The term of
atypical β-AR has emerged to define receptors that display pharmacological properties
different from those of β1-, β2- and β3-AR (Molenaar, 2003). These atypical β-AR are
proposed as distinct states of the β1-AR, in particular a low affinity state (l.a.s.β1-AR)
(Konkar et al., 2000a; Konkar et al., 2000b; Lowe et al., 2002). Therefore, CGP 12177 is
referred to as a non-conventional partial agonist, with agonist properties on β3- and atypical β-
AR, but antagonist on β1- and β2-AR. To further distinguish β3- from atypical β-AR-mediated
responses, other compounds are commonly used, such as the aryloxypropanolamine
derivatives bupranolol (a non-selective β-AR antagonist) and SR 59230A (a selective β3-AR
antagonist), and the phenylethanolamine compounds BRL 37344 and CL 316243 (selective
β3-AR agonists) (Granneman, 2001).
A role of β-AR, distinct from β1- and β2-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. β3-AR agonists were
shown to relax the rat carotid artery (Oriowo, 1994; MacDonald et al., 1999). In rat aorta
precontracted with phenylephrine (PHE), β3-AR agonists induced an endothelium- and NO-
dependent vasorelaxant effect (Trochu et al., 1999). The expression of mRNA and protein of
the β3-AR was recently demonstrated in rat aortic endothelial cells (Rautureau et al., 2002).
The presence of a functional β-AR, presenting more pharmacological similarities with the
atypical β-AR than with the β3-AR, was also reported in rat aorta (Shafiei and Mahmoudian,
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1999; Brawley et al., 2000). However, a recent study provided no evidence for the presence of
functional β3-AR or l.a.s.β1-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 F2α (PGF2α). This study
raises the question of the choice of the preconstrictor agent for investigation of β-AR-
mediated vasorelaxation and suggests a possible interference of some β-AR ligands with the
α-AR signaling pathway (Brahmadevara et al., 2003). The influence of the vasoconstrictor
agent on the responses induced by β3- and atypical β-AR agonists was also noticed in rat
mesenteric artery (Kozlowska et al., 2003). According to these data, it appears that the
presence of functional β3- and atypical β-AR in arteries are still under debate. Moreover, most
of these studies were performed in arteries of systemic circulation, and relative few
information is available in pulmonary circulation. In canine large pulmonary artery, the
existence of a vasorelaxant β3-AR was proposed (Tamaoki et al., 1998). In rat isolated
perfused lung, it was shown that some selective β3-AR agonists (phenylethanolaminotetraline
compounds) opposed the hypoxic vasoconstriction (Dumas et al., 1998). However, the
receptor involved in this effect did not fully match the β3-AR subtype.
The aim of the present study was therefore to evaluate the effect of CGP 12177 and the
other aryloxypropanolamine β-AR ligands, bupranolol and SR 59230A, on the contraction of
isolated rat intralobar pulmonary artery and to investigate a possible interaction of these β-AR
ligands with the α1-AR. For this purpose, the functional responses induced by CGP 12177
were characterized in the absence and presence of α- or β-AR antagonists, and compared to
those of PHE, the reference α1-AR agonist. The affinity of CGP 12177 and several β3- and
atypical β-AR ligands towards the α1-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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Material and Methods
Measurements of isometric tension
Male Wistar rats (450-600 g; 11-18 weeks old; from Elevage Janvier, Le Genest St Isle,
France) and for some experiments female Wistar rats (290-310 g; 15 weeks old; from Elevage
Janvier, Le Genest St Isle, France) were killed by CO2 inhalation. After exsanguination, the
heart and lungs were excised and placed in physiological salt solution (PSS) containing (in
mM): NaCl 119; KCl 4.7; CaCl2 1.5; MgSO4 1.17; KH2PO4 1.18; NaHCO3 25 and glucose
5.5. Intralobar pulmonary arteries (2nd-3rd order branch; internal diameter: 450-850 µm) and,
in some cases, extralobar pulmonary arteries (left and right main branches) were dissected
free of connective tissue. Segments of 1.6 to 2 mm length were mounted in a Mulvany
myograph (Multi Myograph System, model 610M, J.P. Trading, Aarhus, Denmark) and
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 mmHg (Leach et al., 1992). In order 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 (from 10 nM to 100 µM) or the α1-AR agonist PHE (from 1 nM to 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), added 15
min before PHE. In a second procedure, the concentration-response curves to CGP 12177 or
PHE were performed in arteries precontracted with PGF2α (3 µM or 30 µM, concentrations
corresponding approximately to EC15 and EC80, respectively). When indicated, preparations
were incubated with the α-AR antagonist phentolamine (1 µM), bupranolol (5 µM) or SR
59230A (1 or 3 µM) for 25 min before the carrying out of the concentration-response curve to
CGP 12177 or PHE. In order to irreversibly inactivate α-AR, arteries were pre-treated by the
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alkylating agent phenoxybenzamine (PBZ) at 1 µM for 15 min. After PBZ pre-treatment,
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, USA). 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 (in mM): NaCl 118.4; KCl 4.7; CaCl2 2; MgSO4 1.2;
KH2PO4 1.2; NaHCO3 4; Hepes 10 and glucose 6; pH = 7.4 with NaOH) gassed with air and
maintained at 37°C.
Arteries were loaded with fura-PE3 AM (an analogue of fura-2) by incubation in the
microvascular chamber with 1 µM fura-PE3 AM in Krebs-Hepes solution for 90 min at 37°C.
After wash-out with Krebs-Hepes solution, the chamber was placed on the stage of an
inverted epifluorescence microscope (Olympus IX70, 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-PE3 AM was alternately excited at two wavelenghts (345
and 380 nm) selected by a monochromator (Life Science Resources, UK). Digital images
were sampled at 12-bit resolution by a fast scan cooled CDD camera (CoolSNAP fx
Monochrome, Photometrics, 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, USA). Regions of interest were
drawn on the vascular wall to quantify the ratio 345/380, which is an index of the intracellular
calcium concentration. In order 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.
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Arteries were first perfused in the presence of PGF2α (3 µM) during 10 min, and then with
increasing concentrations of CGP 12177 (1 to 100 µM) still in the presence of PGF2α.
[3H]-prazosin binding assay
Brain was rapidly removed from female Wistar rats (180-200 g; 12 weeks old; from Harlan
Interfauna Ibérica, Barcelona, Spain) after decapitation. Cerebral cortex membranes were
prepared as previously described (Madrero et al., 1996). Protein concentration was
determined according to the method of Bradford 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/mmole) 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 (Schleicher and
Schuell, N° 30) 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 ((±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-
2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium), (±)-CGP 12177 hydrochloride
(4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one),
CL 316243 (disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-
1,3-benzodioxole-2,2-dicarboxylate), PGF2α tris salt, phenoxybenzamine hydrochloride,
phentolamine hydrochloride, (-)-phenylephrine hydrochloride, SR 59230A oxalate salt (3-(2-
ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate) and
fura-PE3 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 International (Buckinghamshire, U.K). All drugs were
prepared as stock solution in distilled water, with the exception of SR 59230A and fura-PE3
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AM which were dissolved in dimethylsuphoxide (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-PE3 AM.
Data analysis
Isometric tension is expressed either as percentage of the initial tone (induced by PGF2α 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 values (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 non-linear 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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Results
Influence of the precontractile agent on the response to CGP 12177 in rat pulmonary
arteries
In a first set of experiments, the effect of CGP 12177 was determined in intralobar
pulmonary arteries removed from male rats and precontracted with either 3 µM PGF2α 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 PGF2α compared to 17.5 ± 4.9 % (n=4) with PHE). In
arteries precontracted with 3 µM PGF2α, addition of increasing concentrations of CGP 12177
further enhanced tension in a concentration-dependent manner (Figure 1A&C). In contrast, in
arteries precontracted with 30 nM PHE, CGP 12177 induced a concentration-dependent
relaxation (Figure 1B&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 PGF2α
or 3 µM PHE (inducing 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 PGF2α-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 PGF2α (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
α1-AR in rat pulmonary arteries. In subsequent experiments, the contractile effect of CGP
12177 was systematically compared with the one of PHE, the reference α1-AR agonist.
Influence of tone on the contractile effect of CGP 12177 and PHE in rat intralobar
pulmonary artery
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When applied on the basal tone, CGP 12177 exerted only minor effect in rat intralobar
pulmonary arteries (Figure 2A). By contrast, PHE caused a concentration-dependent
contraction with a pD2 value of 6.3 ± 0.1 (n=23; Figure 2B). Precontraction with PGF2α (3
µM) uncovered the contractile effect of CGP 12177 and markedly enhanced the PHE-induced
contraction (Figure 2A&B). In PGF2α-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.
Effect of CGP 12177 on intracellular calcium concentration in rat intralobar pulmonary
artery
In pressurized intralobar pulmonary arteries loaded with Fura-PE3, PGF2α (3 µM) induced
an increase in intracellular calcium concentration (Figure 3A) and a vasoconstriction (Figure
3B). Addition of CGP 12177 (1 to 100 µM) on PGF2α-precontracted arteries induced a further
increase in both the intracellular calcium concentration (Figure 3A) and contraction (Figure
3B).
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 affected the basal tone (4.7 ± 1.8 % of response to 80 mM KCl (n=8)
compared to 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 to 5.6 ± 0.1 (n=8) in the
presence of CGP 12177; P<0.01), without any changes in the maximal response (Figure 4).
The pKB value of CGP12177 was estimated as 5.3.
Effect of α-AR antagonists on the contractile effect of CGP 12177 and PHE in PGF2α-
precontracted rat intralobar pulmonary artery
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The potential role of α-AR in CGP 12177-induced contractile response was further
investigated using α-AR antagonists. As expected, phentolamine (1 µM), a competitive α-AR
antagonist, did not modify the tension induced by 3 µM PGF2α (tension: 18.0 ± 2.3 % of
response to 80 mM KCl (n=12) in the presence of phentolamine, compared to 14.6 ± 1.2 %
(n=14) in the absence of phentolamine), but significantly shifted to the right the
concentration-response curve to PHE (figure 5B; pD2 = 5.8 ± 0.01 (n=6) in the presence of 1
µM phentolamine, compared to 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 PGF2α-precontracted arteries (Figure 5A). The effect of
another α-AR antagonist, the alkylating agent phenoxybenzamine (PBZ) that irreversibly
inactivates α-AR, was also studied. A 15 min-pretreatment with 1 µM PBZ had no significant
effect on the PGF2α-induced contraction (tension: 19.3 ± 3.0 % of response to 80 mM KCl
(n=8) after PBZ, compared to 14.6 ± 1.2 % (n=14) in the absence of PBZ). PBZ pretreatment
abolished the PHE-induced vasoconstriction in PGF2α-precontracted arteries (Figure 6B) and
markedly reduced the CGP 12177-induced contractile response (Figure 6A).
Effect of atypical β-AR antagonists on the contractile effect of CGP 12177 and PHE in
PGF2α-precontracted rat intralobar pulmonary artery
Bupranolol and SR 59230A are two aryloxypropanolamines commonly used as atypical β-
AR antagonists. At the concentration of 5 µM, bupranolol (a non-selective β-AR antagonist)
did not change the PGF2α-induced tone (tension: 18.0 ± 2.6 % of response to 80 mM KCl
(n=12) in the presence of bupranolol, compared to 14.6 ± 1.2 % (n=14) in the absence of
bupranolol), but significantly decreased the CGP 12177-induced contraction (Figure 7A).
Furthermore, bupranolol caused a rightward shift of the concentration response-curve to PHE
without modifying the maximal response (Figure 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.
In the presence of 3 µM SR 59230A (a drug commonly described as a selective β3-AR
antagonist), the PGF2α-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 to 14.6 ± 1.2
% (n=14) in the absence of SR 59230A; P<0.05) and the contractions induced by CGP 12177
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and PHE were markedly depressed (Figure 8A&B). When the concentration of SR 59230A
was reduced to 1 µM, the PGF2α-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 to
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
(Figure 8A). SR 59230A (1 µM) also caused a significant rightward shift of the PHE
concentration-response curve (Figure 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.
Affinity of different AR ligands for [3H]-prazosin binding sites
The affinities of some α-AR and β-AR ligands towards α1-AR were determined by [3H]-
prazosin binding competition assays. As expected, phentolamine, the reference α-AR
antagonist, completely inhibited the specific binding of [3H]-prazosin (Figure 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
(Figure 9 and Table 1).
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Discussion
The aim of this study was to determine the effect of CGP 12177 and the other
aryloxypropanolamines, bupranolol and SR 59230A, on the contraction of rat pulmonary
artery and the possible interaction of these ligands with α1-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 PGF2α-precontracted
preparations. CGP 12177-induced contraction was inhibited by phentolamine and PBZ (α-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 β-AR agonists. For instance, CGP 12177 produced relaxation in PHE-precontracted rat
aorta, but failed to do so in PGF2α-precontracted one (Brahmadevara et al., 2003). Similarly,
the relaxation evoked by cyanopindolol (another aryloxypropanolamine, non-conventional
partial agonist) in rat mesenteric artery was strongly reduced in vessels constricted with
PGF2α, compared to those precontracted with PHE (Kozlowska et al., 2003). In the present
study, the effect of CGP 12177 was also evaluated in arteries precontracted with PGF2α
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 PGF2α (producing only 15 % of its
maximum response) might explain why contractile effects of CGP 12177 could be unmasked
here. Accordingly, increasing pre-contraction level resulted in a marked reduction in the
amplitude of CGP 12177-induced contraction.
Interference of CGP 12177 with α-AR or α-AR-mediated intracellular signaling pathway
has been postulated from experiments showing that CGP 12177 produced relaxation in PHE-
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precontracted rat aorta, but not in those precontracted with PGF2α (Brahmadevara et al.,
2003). The present study provides evidence for an interaction of CGP 12177 at the level of
α1-AR. Our experimental data support the idea that α1-AR agonist properties contributed to
the contractile effect of CGP 12177 in PGF2α-precontracted arteries. The increase in
intracellular calcium concentration evoked by CGP 12177 is consistent with α1-AR-mediated
response. Moreover, CGP 12177-induced contraction was diminished by phentolamine and
PBZ. Since the latter compounds are non-selective α-AR antagonists, a potential contribution
of α2-AR in the effect of CGP 12177 cannot be excluded. Both phentolamine and PBZ also
inhibited the effect of the selective α1-AR agonist PHE. With phentolamine, an apparent
competitive antagonism was obtained and its estimated pKB value (7.9) is consistent with the
affinity for α1-AR determined in the present study (pKi value 7.8) and in literature (Yan et al.,
2001). Eventhough 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 α-AR antagonist PBZ, PHE-induced contraction was abolished, while CGP
12177-induced contraction was partially inhibited. If the effect of CGP 12177 was exclusively
due to the activation of α-AR, it would be expected to be also totally abolished by PBZ. Thus,
CGP 12177-induced contraction can be attributed to α-AR activation and to an α-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 PGF2α) seems a
priori not consistent with α1-AR agonist properties of this compound. However, in conditions
that unmasked the contractile properties of CGP 12177, PGF2α increased the sensitivity to
PHE. Different mechanisms may underlie PGF2α-induced potentiation of α1-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 α-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 ERK-MAP kinase cascade (Bhattacharya and Roberts,
2003). The implication of these pathways in enhanced sensitivity to PHE in PGF2α-
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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 PGF2α could be
related to a low intrinsic efficacy of CGP 12177 on α1-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 α1-AR. In
conditions in which it had only 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 α1-AR. Competition for [3H]-prazosin binding sites clearly
demonstrated the α1-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 α1-AR in the
effect of CGP 12177 in rat pulmonary artery.
In this study, other aryloxypropanolamines were evaluated as potential α-AR ligands.
Recently, it has been reported that bupranolol (a non-selective β-AR antagonist) and SR
59230A (a selective β3-AR antagonist) elicited relaxation in PHE-precontracted rat aorta, but
not in PGF2α-precontracted one (Brahmadevara et al., 2003). This suggests that, like CGP
12177, bupranolol and SR 59230A interfere with the α1-AR signaling pathway
(Brahmadevara et al., 2003). The present study shows that bupranolol and SR 59230A both
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 α1-AR. At concentrations > 1 µM, SR 59230A
markedly depressed the maximal response to PHE. Thus, additional mechanisms to α-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 β3-AR and/or atypical β-AR
(Horinouchi and Koike, 2001). However, it is unlikely that such β3-AR and/or atypical β-AR
agonists properties accounted for the antagonist effect of SR 59230A seen here since SR
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59230A did not antagonize, but enhanced PGF2α-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 α1-AR in
the effect of CGP 12177 in pulmonary artery. The relative contribution of α1-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 α1-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 α1-AR by CL 316243 and BRL 37344, owing to
the low binding affinity of these two latter agents for α1-AR. CL 316243 and BRL 37344 are
phenylethanolamine derivatives, whereas CGP 12177, bupranolol and SR 59230A are
aryloxypropanolamine ones (Nisoli et al., 1996; Strosberg, 1997). Aryloxypropanolamines
likely share structural features responsible for interaction with α-AR. A detailed structure-
activity relationship would be necessary to clarify the molecular requirements for
concomitantly binding to α-AR and β-AR. Nevertheless, phenylethanolamine derivatives
appear more suitable tools than aryloxypropanolamines to investigate the role of β3-AR in the
vasculature.
In conclusion, this study gives more insight into the pharmacological properties of several
β-AR ligands. It shows the affinity of CGP 12177, bupranolol and SR 59230A for α1-AR. It
also indicates that bupranolol and SR 59230A display antagonist effect towards α1-AR in rat
pulmonary artery. Finally, it demonstrates that in this artery, CGP 12177 exhibits partial α1-
AR agonist activity. Aryloxypropanolamine compounds should be used with caution to
investigate the role of β3- and atypical β-AR in the regulation of vascular contraction.
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Acknowledgments
We thank Martine Lacayrerie for excellent animal care. We also thank Nadège Bellance and
Le Van Nha Phuong for helpful contribution in contractile experiments.
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Footnotes
a) 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).
b) Address for reprint requests:
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
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Legends for figures
Figure 1
Effect of CGP 12177 in rat intralobar pulmonary arteries precontracted with either 3 µM
PGF2α or 30 nM PHE. A & B: typical recordings. C: concentration-response curves
representing the mean ± S.E.M. of 4-8 experiments. The response is expressed as the
percentage of pre-contraction, 100% corresponding to the contraction induced by either
PGF2α or PHE. Asteriks denoted statistical significance between concentration-response
curves, as determined by a two-way analysis of variance (*** P < 0.001).
Figure 2
Effect of CGP 12177 (A) and PHE (B) on the basal tone of rat intralobar pulmonary arteries or
after precontraction with 3 µM PGF2α. Each point is the mean ± S.E.M. of 5-23 experiments.
The response is expressed as percentage of the contraction induced by 80 mM KCl. The
legend of the Y axis is identical for Panels A and B. Asteriks denoted statistical significance
between concentration-response curves, as determined by a two-way analysis of variance (***
P < 0.001).
Figure 3
Simultaneous effect of CGP 12177 on intracellular calcium concentration (expressed as the
ratio of 345/380 signals, Panel A) and contraction (expressed as external diameter recording,
Panel B) in pressurized rat intralobar pulmonary arteries, loaded with Fura-PE3 and pre-
incubated with 3 µM PGF2α. Representative traces out of 3 independent experiments.
Figure 4
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Effect of PHE, in the absence or presence of 100 µM CGP 12177, in rat intralobar pulmonary
arteries. Each point is the mean ± S.E.M. of 7-8 experiments. The response is expressed as
percentage of the contraction induced by 80 mM KCl. Asteriks denoted statistical significance
between concentration-response curves, as determined by a two-way analysis of variance (**
P < 0.01). For statistical analysis, the increase in basal tone observed during the pretreatment
has been substracted.
Figure 5
Effect of CGP 12177 (A) and PHE (B), in the absence or presence of 1 µM phentolamine, in
rat intralobar pulmonary arteries precontracted with 3 µM PGF2α. Each point is the mean ±
S.E.M. of 6-8 experiments. The response is expressed as percentage of the contraction
induced by PGF2α. The legend of the Y axis is identical for Panels A and B. Asteriks denoted
statistical significance between concentration-response curves, as determined by a two-way
analysis of variance (*** P < 0.001).
Figure 6
Effect of CGP 12177 (A) and PHE (B) in rat intralobar pulmonary arteries pretreated or not
with 1 µM PBZ and precontracted with 3 µM PGF2α. Each point is the mean ± S.E.M. of 3-6
experiments. The response is expressed as percentage of the contraction induced by PGF2α.
The legend of the Y axis is identical for Panels A and B. Asteriks denoted statistical
significance between concentration-response curves, as determined by a two-way analysis of
variance (*** P < 0.001).
Figure 7
Effect of CGP 12177 (A) and PHE (B), in the absence or presence of 5 µM bupranolol, in rat
intralobar pulmonary arteries precontracted with 3 µM PGF2α. Each point is the mean ±
S.E.M. of 6-8 experiments. The response is expressed as percentage of the contraction
induced by PGF2α. The legend of the Y axis is identical for Panels A and B. Asteriks denoted
statistical significance between concentration-response curves, as determined by a two-way
analysis of variance (** P < 0.01; *** P < 0.001).
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Figure 8
Effect of CGP 12177 (A) and PHE (B), in the absence or presence of 1 or 3 µM SR 59230A,
in rat intralobar pulmonary arteries precontracted with 3 µM PGF2α. Each point is the mean ±
S.E.M. of 4-8 experiments. The response is expressed as percentage of the contraction
induced by PGF2α. The legend of the Y axis is identical for Panels A and B. Asteriks denoted
statistical significance between concentration-response curves, as determined by a two-way
analysis of variance (* P < 0.05; *** P < 0.001).
Figure 9
Displacement of specific [3H]-prazosin binding in rat cerebral cortex membranes by
phentolamine, SR 59230A, bupranolol, CGP 12177, BRL 37344 and CL 316243. Each point
is the mean ± S.E.M. of 3-6 experiments.
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Figure 1
BCGP 12177
A C
50
100
150
200
250
300
-log [CGP 12177] (M)
8 7 6 5 4C
ontr
actil
e re
spon
se(%
pre
cont
ract
ion)
***
PGF2α-precontracted
PHE-precontracted
CGP 121770.01 0.1 1 10 100 µM
10 min
0.2
mN
/mm
PGF2α
PHE
10 min
0.2
mN
/mm
0.01 0.1 1 10 100 µM
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-log [CGP 12177] (M)
8 7 6 5 4
Con
trac
tile
resp
onse
(% 8
0 m
M K
Cl)
0
10
20
30
40
50
A B
Figure 2
0
10
20
30
40
50
-log [PHE] (M)8 7 6 5 49
***
PGF2α-precontracted
Basal tone
***
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Ext
erna
l dia
met
er(µ
m)
10010
1
8 min
Rat
io(3
45/3
80 n
m)
540
560
580
600
620
1
1.2
1.4
1.6
1.8
2
Figure 3
A
B
µMCGP 12177
PGF2α
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100 µM CGP 12177
-log [PHE] (M)
Con
trac
tile
resp
onse
(% 8
0 m
M K
Cl) **
0
10
20
30
40
50
8 7 6 5 49
Figure 4
Control
JPET #61192
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100
150
200
250
300
-log [CGP 12177] (M)8 7 6 5 4
Con
trac
tile
resp
onse
(% P
GF
2α)
100
200
300
400
500
-log [PHE] (M)8 7 6 5 49
Control1 µM Phentolamine
A B
Figure 5
***
***
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B
Con
trac
tile
resp
onse
(% P
GF
2α)
ControlPBZ pretreatment
Figure 6
100
200
300
400
500
-log [PHE] (M)
8 7 6 5 49
***
-log [CGP 12177] (M)
8 7 6 5 4
A
100
150
200
250
300
***
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Figure 7
100
200
300
400
500
-log [PHE] (M)
8 7 6 5 49
A B
-log [CGP 12177] (M)
8 7 6 5 4
100
150
200
250
300
Con
trac
tile
resp
onse
(% P
GF
2α)
Control5 µM Bupranolol
*****
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Figure 8
100
200
300
400
500
-log [PHE] (M)
8 7 6 5 49
A B
-log [CGP 12177] (M)
8 7 6 5 4
100
150
200
250
300
Con
trac
tile
resp
onse
(% P
GF
2α)
Control1 µM SR 59230A
3 µM SR 59230A
***
*
***
*
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0
20
40
60
80
100
-log [competitor agent] (M)
[3 H]-
praz
osin
bin
ding
(%)
Figure 9
9 7 6 5 4 3811
CGP 12177
CL 316243
SR 59230A
BRL 37344
Bupranolol
Phentolamine
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JPET #61192
1
Table 1 : Affinity of phentolamine and ß-AR ligands for α1-AR.
pKi values were determined by displacement of [3H]-prazosin binding in rat cerebral cortex
membranes.
pKi
(or % inhibition at 1 mM)
Phentolamine 7.78 ± 0.07 (n=4)
SR 59230A 6.61 ± 0.11 (n=4)
Bupranolol 5.73 ± 0.01 (n=3)
CGP 12177 5.22 ± 0.07 (n=6)
BRL 37344 3.75 ± 0.24 (n=4)
CL 316243 N.D. (n=3) (18.4 ± 3.8)§
§ When pKi values could not be determined, % inhibition of [3H]-prazosin binding obtained
with 1 mM competitor agent is given in brackets.
Data are means ± S.E.M. of n separate experiments. N.D.: not determined.
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