© 1995 American Heart Association, Inc.
Articles |
Binding of A1 Adenosine Receptor Ligand [3H]8-Cyclopentyl-1,3-Dipropylxanthine in Coronary Smooth Muscle
From the Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC.
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Abstract |
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Abstract Vascular smooth muscle has been reported to contain the A1 subtype of adenosine receptors, but the existence of such receptor(s) in coronary smooth muscle has not been established. In the present study, the 3H-labeled A1-selective antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) was used to demonstrate the specific binding in porcine coronary artery smooth muscle membranes. The binding was saturable with a Bmax of 6.43±1.02 fmol/mg protein. Scatchard analysis of the binding data provided a single binding site with a Kd of 0.21±0.025 nmol/L. In the competition experiments, adenosine receptor agonists and antagonists showed the following order of potency (nmol/L): S-N6-(2-endo-norbornyl)adenosine (S-ENBA) 0.11=R(-)-N6-phenylisopropyladenosine 0.32>DPCPX 3.2=xanthine amine congener 2.4=N6-cyclopentyladenosine 2.67>5'-(N-ethylcarboxamido)-adenosine 7.35>>2-[p-(2-carboxyethyl)-phenethyl-amino]-5'-(N-ethylcarboxamido)-adenosine 1000>theophylline 83 000. This order of potency fits the criteria for the A1 adenosine receptor. S-ENBA, a highly selective A1 receptor agonist, was used to investigate the effect on isoproterenol-mediated vasorelaxation and cAMP accumulation. S-ENBA (0.1 to 10 nmol/L) dose-dependently shifted the isoproterenol-mediated (10-8 to 10-5 mol/L) vasorelaxation to the right in vascular rings. S-ENBA (10 nmol/L) inhibited the basal cAMP levels by 36% and attenuated the isoproterenol (10-5 mol/L)–stimulated cAMP by 25% in the coronary rings. These inhibitory effects of S-ENBA on isoproterenol-mediated cAMP-accumulation and vasorelaxation were abolished by pertussis toxin (100 ng/mL, overnight) treatment of the arteries. Similarly, the effects of S-ENBA on isoproterenol-mediated responses were antagonized by DPCPX. This study provides the evidence suggesting the existence of A1 adenosine receptors linked to adenylyl cyclase in an inhibitory manner through pertussis toxin–sensitive G proteins in the coronary artery.
Key Words: A1 adenosine receptor • coronary artery • adenosine receptor antagonist • cAMP • G proteins
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adenosine is a ubiquitous modulator of various physiological processes.1 Several specific adenosine receptor subtypes (A1, A2a, A2b, and A3) have been cloned recently.2 These receptor subtypes can be distinguished by the stimulation of their second messengers and structure-activity relation of various adenosine receptor agonists and antagonists.3 4 Vascular smooth muscle is believed to contain the A2 subtype of adenosine receptor, which is linked to adenylyl cyclase in a stimulatory manner via G proteins, producing vasorelaxation.5 6 7 Recently, however, certain reports8 9 10 11 12 suggest the coexistence of A1 and A2 receptor subtypes in vascular smooth muscle, including the coronary artery. An A1 receptor has been reported to be linked to guanylyl cyclase in smooth muscle cells isolated from rat and calf aortas and bovine coronary arteries9 and to K+ channels in porcine coronary arteries,10 mediating vasorelaxation. Others11 have reported an A1 receptor linked to adenylyl cyclase in an inhibitory manner in porcine coronary smooth muscle cells, which did not seem to be a relaxing receptor. Furthermore, a vasoconstrictive response in porcine coronary artery has been demonstrated by N6-cyclopentyladenosine (CPA, an A1-selective agonist) and not by other adenosine analogues.12 Therefore, there exists a great deal of controversy as to the existence and function of such receptors, which lack the characterization provided for adenosine receptors in the coronary artery by using selective radioligand binding studies. In the present study, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1-selective antagonist, was used as a 3H-labeled radioligand to demonstrate a specific receptor binding in porcine coronary arterial smooth muscle membranes. Furthermore, the functional response of A1-selective agonists on cAMP was also examined in the present study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Membrane Preparation
Pigs were killed at a local slaughterhouse, and within 30 minutes their hearts were obtained and transported to the laboratory in cold Krebs-Henseleit buffer (pH 7.4). Left anterior descending and circumflex coronary arteries were dissected from the hearts. The arteries were cleaned of fat and connective tissue. Endothelial cells were removed by cutting the arteries longitudinally and gently scraping the intimal surface with a scalpel blade. Arteries were quickly frozen in liquid nitrogen. The frozen arteries were pulverized and homogenized in Tris-HCl buffer (pH 7.4) containing (mmol/L) sucrose 250, EDTA 5, and polymethylsulfonyl fluoride 2. The homogenate was filtered through three layers of cotton gauze. The filtrate was centrifuged at 900g for 10 minutes and finally at 105 000g for 60 minutes. The pellet was suspended in the same buffer but with no sucrose and stored at -70°C until use. The protein was determined according to the procedure described by Bradford,13 with
-globulin used as a standard.
[3H]DPCPX Binding Assay
Binding of [3H]DPCPX to porcine coronary artery
membranes was performed essentially according to the method described
by Lohse et al.14 The reaction mixture in a volume of 250
µL contained 50 mmol/L Tris-HCl buffer (pH 7.4), 1 mmol/L
Mg2+, and 200 µg membrane protein
pretreated with 0.2 U/mL adenosine deaminase at 25°C. The ligand
concentration varied from 0.02 to 2 nmol/L. The reaction lasted for 2
hours at 25°C with a gentle shaking. Reaction was terminated by a
rapid filtration followed by three washes (4 mL each) of ice-cold
incubation buffer through Whatman GF/B glass fiber filters under
vacuum by use of a Brendel harvester. The filters were presoaked in the
incubation buffer containing 0.3% polyethylenimine for 2 hours. The
radioactivity on the filters was determined by liquid scintillation
counting for 5 minutes at an efficiency of 
60%. The nonspecific
binding was defined by the addition of 100 µmol/L
R(-)-N6-phenylisopropyladenosine
(R-PIA). All assays were performed in triplicate.
In competition experiments, various competitors were added in a range
of 10-11 to 10-4 mol/L with a fixed
concentration (0.3 nmol/L) of [3H]DPCPX while
maintaining assay conditions similar to those described above.
Organ Bath Experiments and cAMP Accumulation
The functional response of A1 receptor activation on
vascular tone and cAMP accumulation was investigated in isolated
coronary rings. Briefly, porcine left anterior descending and
circumflex arteries were cut into 3- to 4-mm-wide rings. Endothelium
was removed by rubbing the intimal surface, and the rings were mounted
in 10-mL organ baths filled with Krebs-Henseleit buffer (pH 7.4). The
baths were supplied with 95% O2/5% CO2
and maintained at 37°C. Changes in isometric tension were measured
with force transducers (Grass FT03) connected to Sensormedics
dynographs (model R611). The vascular rings were equilibrated at an
initial tension of 2 g for 1 hour, and the buffer was changed every 15
minutes. Thereafter, the rings were challenged with 35 mmol/L KCl until
constant and reproducible contractions were achieved. The absence of
endothelium was tested by the inability of bradykinin (0.1 µmol/L) to
relax the 35 mmol/L KCl–contracted vascular rings.
After sustained and reproducible contractions with 35 mmol/L KCl were
achieved, isoproterenol (10-8 to 10-5 mol/L)
was added in a cumulative fashion in the absence and presence of 0.1,
1.0, and 10 nmol/L
S-N6-(2-endo-norbornyl)adenosine
(S-ENBA) to the bath to obtain concentration response–relaxation
curves. The addition of S-ENBA was made 5 minutes before isoproterenol
administration. The loss in tone due to isoproterenol in 35 mmol/L
KCl–contracted rings was calculated as percent relaxation. To
investigate the role of G proteins on S-ENBA–mediated response, the
arteries were treated with pertussis toxin (PTx, 100 ng/mL). The
following day, isoproterenol (10-8 to 10-5
mol/L)–mediated relaxation in the absence and presence of 10 nmol/L
S-ENBA (the most effective dose; see Fig 2
) was studied in both
(control and PTx-treated) groups. Furthermore, to determine the
specificity of S-ENBA, the effects of S-ENBA on isoproterenol were also
investigated in the presence of A1 receptor antagonist
DPCPX at a concentration of 50 nmol/L. Other experimental conditions
remained the same as described above.
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cAMP was determined as described in an earlier report from this
laboratory.15 Vascular rings from control and PTx-treated
(100 ng/mL) arteries were equilibrated in Krebs' buffer for 1 hour,
with a change to fresh buffer every 15 minutes. The rings were
challenged with 35 mmol/L KCl for an additional hour by changing the
buffer and adding 35 mmol/L KCl. These conditions were similar to those
in organ bath studies. Twenty minutes after the last addition of KCl
(35 mmol/L), S-ENBA (10 nmol/L), isoproterenol (10-5
mol/L), and S-ENBA+isoproterenol were added and incubated for 20
minutes. In another set of experiments, the rings were also incubated
with 50 nmol/L DPCPX for 30 minutes before adding the agonists. Rings
without agonist served as control (basal). The rings were quickly
frozen in liquid nitrogen, pulverized, and homogenized in 50 mmol/L
acetate buffer, pH 6.2, containing 100 µmol/L papaverine. Part of the
homogenate was used for protein estimation; the remainder was heated
for 3 minutes in a boiling water bath for cAMP measurement.
Radioimmunoassay for cAMP was performed by the method described by
Harper and Brooker16 and adopted by us
earlier.15 The samples were acetylated with acetic
anhydride and triethylamine, and then 125I-cAMP (5000
cpm) and cAMP antisera were added and gently mixed. After 18 hours of
incubation in a refrigerator, 125I-cAMP (bound) was
separated (from free) and counted in a gamma counter (Packard 5000).
The results were expressed as femtomoles cAMP per milligram
protein.
Chemicals
[3H]DPCPX (120 Ci/mmol) and 125I-cAMP
were purchased from New England Nuclear. S-ENBA, R-PIA, CPA, xanthine
amine congener (XAC), DPCPX,
5'-(N-ethylcarboxamido)-adenosine (NECA), and
2-[p-(2-carboxyethyl)-phenethyl-amino]-5'-(N-ethylcarboxamido)-adenosine
(CGS 21680) were purchased from Research Biochemicals International.
cAMP antibody was purchased from Kew Scientific. PTx was purchased from
List Biologicals Inc. All other chemicals of the highest purity
available were purchased from either Sigma Chemical Co or Fisher
Scientific Co.
Statistical Analysis
The statistical significance between the groups was evaluated by
Student's t test. A value of P<.05 was
considered significantly different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[3H]DPCPX Binding
A saturation isotherm of [3H]DPCPX binding in porcine coronary artery membranes is shown in Fig 1
.
[3H]DPCPX binding was saturable with a low binding
capacity of 6.43±1.02 fmol/mg protein. Scatchard transformation of the
data provided a linear plot indicating a homogenous population of
binding sites with a dissociation constant (Kd)
of 0.21±0.025 nmol/L. Specific binding at Kd
was 40% of the total binding.
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Various agonists and antagonists competed [3H]DPCPX
binding (Table 1). The A1
receptor–selective agonists competed the binding with high affinity:
S-ENBA=R-PIA>CPA. Similarly, DPCPX, an A1
receptor–selective antagonist (DPCPX=XAC), competed
[3H]DPCPX binding with high affinity. NECA, a nonelective
analogue, competed with the binding at a lower affinity, whereas CGS
21680, an A2a-selective analogue, was a poor competitor of
[3H]DPCPX binding. Theophylline was found to be a very
weak competitor of [3H]DPCPX binding.
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Organ Bath Experiments and cAMP Accumulation
Since this and other4 studies show S-ENBA as a highly
selective A1 adenosine receptor agonist, its effect was
examined on the ß-receptor agonist (isoproterenol)–mediated
relaxation and cAMP accumulation in porcine coronary arterial rings.
Fig 2
, top, demonstrates a significant rightward shift
in isoproterenol (10-8 to 10-5
mol/L)–mediated relaxation in the presence of various concentrations
of S-ENBA (0.1, 1.0, and 10 nmol/L). However, S-ENBA at 0.1 nmol/L was
unable to affect isoproterenol-mediated relaxation, whereas 1 and 10
nmol/L significantly (P<.05) attenuated the relaxation. It
should be noted that S-ENBA alone at 1 to 10 nmol/L did not affect the
tone of vascular rings.
Since S-ENBA at 10 nmol/L produced a pronounced rightward shift in
isoproterenol-mediated relaxation, this concentration of S-ENBA was
used to test the effect on basal and 10-5 mol/L
isoproterenol-induced accumulation of cAMP. Table 2
shows the experimental data. S-ENBA (10 nmol/L) attenuated
isoproterenol-stimulated cAMP by 25%. S-ENBA (10 nmol/L) and
isoproterenol (10-5 mol/L), respectively, inhibited (36%)
and stimulated (46%) cAMP over the basal level. The cAMP level without
any agonist was represented as the basal value.
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PTx (100 ng/mL, overnight) treatment of the arteries abolished the
effects of S-ENBA (10 nmol/L) on isoproterenol-mediated relaxation (Fig 2, bottom). Such PTx treatment itself did not affect the
isoproterenol-mediated relaxation curve compared with the curve for the
untreated arteries (data not shown). Similarly, the inhibitory effects
of S-ENBA (10 nmol/L) on basal as well as isoproterenol-stimulated cAMP
accumulation were found to be reversed by PTx treatment. The basal and
isoproterenol-stimulated cAMP levels were not significantly different
in toxin-treated arteries compared with control arteries (Table 2).
Also, the A1 receptor antagonist DPCPX (50 nmol/L)
antagonized the inhibitory effects of 10 nmol/L S-ENBA on
isoproterenol-mediated relaxation as well as cAMP accumulation (Fig 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrated a specific receptor-ligand binding using [3H]DPCPX, an A1 selective antagonist, to characterize adenosine receptors in porcine coronary arterial smooth muscle membranes. The binding was found to be saturable, with a binding capacity of 6.43±1.02 fmol/mg protein, suggesting a low receptor population. Scatchard analysis of the data provided a linear plot with a Kd of 0.21±0.025 nmol/L. Furthermore, nanomolar concentrations of S-ENBA, an A1-selective agonist, attenuated vasorelaxation and cAMP stimulation mediated by ß-receptor agonists in porcine coronary arterial rings. This is the first report showing specific receptor binding along with the functional response of A1 adenosine receptors in the coronary artery.
[3H]DPCPX has been extensively used as a ligand for the characterization of A1 adenosine receptors in a variety of tissues.14 17 18 The affinity constant of [3H]DPCPX for A1 receptors ranges from 0.2 to 0.5 nmol/L in rat striatum,14 ventricular myocytes,17 and bovine testes.18 These studies support our observation that [3H]DPCPX binds to porcine coronary membranes with a high affinity (Kd=0.21±0.025 nmol/L). The pharmacological profile of various agonists and antagonists to adenosine receptors was established. A1-selective agonists like S-ENBA, R-PIA, and CPA and antagonists like DPCPX and XAC were found to be more potent than nonselective NECA and the A2a-selective agonist CGS 21680 in displacing the [3H]DPCPX binding. The high affinity of DPCPX and XAC and low affinity of CGS 21680 in the present study ruled out the possibilities of A3 and A2a receptors. Xanthine analogues are known to have poor affinity toward A3 receptors,2 whereas CGS 21680 binds with a high affinity (10 to 15 nmol/L) to A2a receptors.2 This potency profile (R-PIA>NECA>CGS 21680) is a better fit of the criteria for A1 adenosine receptor reported in several tissues.3 4
Historically, coronary arterial smooth muscle has been known to contain A2 vasorelaxing adenosine receptors.5 19 However, several reports now suggest that there are adenosine receptors other than A2 involved in vasorelaxation. Kurtz9 and Dart and Standen10 have suggested that the coronary artery contains A1 receptors that produce vasorelaxation through guanylyl cyclase and K+ channel activation, respectively. Merkel et al12 have shown that glibenclamide, a K+ channel antagonist, can block the relaxation mediated by an A1-selective agonist (CPA) but not an A2-selective agonist (N6-[2-(3,5-dimethoxyphenyl)2-(2-methylphenyl)-ethyl]adenosine). They allowed for the possibility of a receptor, other than the typical A1 and A2a receptors, linked to the K+ channel and mediating vasorelaxation. Later, on the basis of electrophysiological observations, a putative A4 adenosine receptor linked to the K+ channel and causing vasorelaxation was proposed to exist in porcine coronary artery.20 In the same study, rat striatum was also reported to contain A4 receptors. Recently, however, this laboratory did not find the involvement of glibenclamide-sensitive ATP-sensitive K+ channels in adenosine receptor–mediated relaxation of the porcine coronary artery.21 All these studies have added a new dimension toward understanding the nature and function of coronary adenosine receptors.
In the present study, the receptor determined by
[3H]DPCPX binding does not seem to be a vasorelaxing
adenosine receptor. We found that the A1-selective agonist
S-ENBA significantly attenuated the relaxation mediated by
isoproterenol. Similar attenuation in isoproterenol-mediated relaxation
was also found with nanomolar concentrations of other
A1-selective agonists (CPA and R-PIA; data not shown). The
mechanism of attenuation appeared to be through the inhibition of
adenylyl cyclase via Gi proteins after the activation of
the A1 receptor by the agonist. This notion is supported by
our cAMP data (Table 2). S-ENBA inhibited the basal and
isoproterenol-stimulated cAMP accumulation in the coronary rings. These
effects of S-ENBA on cAMP were found to be abolished when the arteries
were treated with PTx. Similarly, the inhibitory effect of S-ENBA on
isoproterenol-mediated relaxation was also reversed in the
toxin-treated arteries. This suggests that the effects of S-ENBA in the
coronary artery are mediated through a PTx-sensitive G protein.
Treatment of the tissues with PTx has been shown to uncouple the
receptors from the Gi protein (an inhibitory G protein
linked to adenylate cyclase) and attenuate the receptor
agonist–mediated cellular responses.22 23 24 Furthermore,
the existence of a PTx-sensitive 41-kD protein such as
Gi
in porcine and human coronary arterial smooth muscle
membranes has been reported previously by us.23 25 Mills
and Gewirtz11 have reported the involvement of a G protein
in the inhibition of adenylyl cyclase by A1-selective
agonists in porcine coronary smooth muscle cultured cells, which
supports our observation. However, apart from adenylyl cyclase, other
effector systems including the activation of ATP- and
acetylcholine-sensitive K+ channels, changes in inositol
phosphates, and the inactivation of Ca2+ channels
have been shown to mediate the cellular responses of A1
receptors in a variety of tissues.3 The responses of most
of these effectors to A1 receptors were found to be
dependent on the stimulation of G proteins. Furthermore, we observed
that DPCPX was able to antagonize the inhibitory effects of S-ENBA on
isoproterenol-mediated responses, suggesting specific A1
receptor involvement.
On the basis of the present and other studies,8 9 10 11 12 it seems that there is more than one type of A1 adenosine receptor linked to different effectors and/or the same receptor is linked to different effectors in more than one affinity state in the coronary artery. Nevertheless, the receptor we report in the present study seems to be an inhibitory A1 adenosine receptor linked to adenylyl cyclase. The role of such an inhibitory adenosine receptor in the coronary artery remains to be established. Despite the observation that the A1-selective agonist was able to inhibit the basal and isoproterenol-stimulated cAMP as well as to attenuate the relaxation mediated by isoproterenol, we did not observe a vasocontractile response to S-ENBA or to other A1 agonists alone. Possibly, such an inhibition of adenylyl cyclase by the A1 receptor agonist S-ENBA could not generate enough force to produce contraction in the coronary artery but was enough to oppose the relaxation mediated by isoproterenol. However, such an inhibitory A1 receptor may be important in modulating the vascular tone under pathophysiological conditions in which the receptor is overexpressed. This speculation needs to be investigated.
In summary, this is the first study demonstrating that porcine coronary arterial smooth muscle contains a saturable and displaceable binding site characterized as the A1 receptor. The cAMP data suggest that this receptor may be linked to adenylyl cyclase in an inhibitory manner through PTx-sensitive G proteins.
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Acknowledgments |
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This study was supported by National Heart, Lung, and Blood Institute grant HL-27339 and the American Heart Association, North Carolina Affiliate, Inc.
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Footnotes |
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Reprint requests to S. Jamal Mustafa, Professor, Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27858.
Received September 12, 1994; accepted March 28, 1995.
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