© 1995 American Heart Association, Inc.
Articles |
Adenosine A2a and A2b Receptors in Cultured Fetal Chick Heart Cells
High- and Low-Affinity Coupling to Stimulation of Myocyte Contractility and cAMP Accumulation
From the Department of Medicine, Cardiovascular Division, and the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia.
Correspondence to Bruce T. Liang, MD, 504 Johnson Pavilion, University of Pennsylvania Medical Center, 36th & Hamilton Walk, Philadelphia, PA 19104-6060.
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Abstract Adenosine exerts pronounced biological effects in the heart cell. The role of multiple adenosine receptor subtypes in regulating the heart cell function is not known. Ventricular cells cultured from chick embryos 14 days in ovo were used to study a novel feature of heart cell regulation by the stimulatory adenosine receptors. The inhibitory adenosine A1 receptor pathway was first inactivated by pertussis toxin treatment of the cultures, and the effects of adenosine agonists and antagonists on the heart cell contractile amplitude, measured via an opticovideo motion detection system, and on the modulation of cAMP level were determined. Adenosine and N-ethyladenosine-5'-uronic acid (NECA), capable of activating both the adenosine A2a and A2b receptors, caused a greater increase in the contractile amplitude than did the A2a-selective agonist 2-[4-(2-carboxyethyl)phenylethylamino]-5'- N-ethylcarboxamidoadenosine (CGS21680). NECA caused a biphasic increase in cAMP, which became monophasic in the presence of the A2a receptor–selective antagonist 8-(3-chlorostyryl)caffeine, whereas the CGS21680-induced cAMP response was monophasic. Blocking with 8-(3-chlorostyryl)caffeine abolished most of the CGS21680-elicited contractile or cAMP response while attenuating only part of the adenosine- or NECA-stimulated responses. Blocking with the A2b-selective antagonists 1,3-diethyl-8-phenylxanthine or alloxazine caused a more pronounced inhibition of the contractile or cAMP response by adenosine or NECA than by CGS21680. Affinity of the A2a receptor was 60-fold higher than that of the A2b receptor. These data demonstrate that a functional A2b receptor is expressed on the heart cell and is capable of mediating augmentation of cardiac myocyte contractility and that adenosine A2a and A2b receptors, with greatly different affinity, coexist and are coupled to the same functional responses. Taken together, the data suggest a novel feature of heart cell regulation, where the high-affinity A2a receptor can play an important modulatory role in the presence of a low level of adenosine, whereas the low-affinity A2b receptor becomes functionally important when the adenosine level is high.
Key Words: adenosine receptors • cultured heart cells
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although subtypes of the same class of receptor, such as ß-adrenergic and
-adrenergic receptors,
coexist in the heart, they are coupled to different effectors and
mediate separate and distinct functions.1 2 3 4 5 6 7 8 9 Whether
receptor subtypes with different affinity for the same agonist can
couple to the same functional response in the same cardiac cells is not
clear. Adenosine is an important regulatory metabolite that exerts
pronounced biological effects in the cardiovascular
system10 11 12 13 that are mediated by cell-surface adenosine
receptors. In studies of a stimulatory adenosine action in cultured
fetal chick heart cells, adenosine and adenosine agonists were capable
of causing a significant increase in myocyte
contractility,14 indicating the involvement of stimulatory
adenosine receptors. Since adenosine A2a and
A2b receptors, both representing stimulatory
adenosine receptors, have greatly different affinity for their agonist,
adenosine,15 16 17 and since the adenosine
agonist–stimulated cardiac cAMP formation occurred with
EC50 >1 µmol/L in studies demonstrating such a
stimulatory effect,14 18 19 20 21 22 the question arises regarding
whether A2a and A2b subtypes are both expressed
in the heart cell and are capable of mediating augmentation of myocyte
contractility and stimulation of cAMP accumulation. Studies of this
question will determine whether a novel feature of cardiac cell
regulation exists in which the agonist adenosine and its high-affinity
(A2a) and low-affinity (A2b) receptors can
serve dual modulatory roles. Under conditions in which there is a low
level of adenosine, the A2a receptor may serve an important
regulatory role, whereas under conditions of hypoxia or ischemia, in
which there is a large amount of adenosine released, the
A2b receptor can become functionally significant. Using atrial and ventricular myocytes cultured from chick embryos 14 days in ovo, we have previously shown that a stimulatory adenosine receptor, likely the adenosine A2 receptor, was present on the ventricular but not the atrial myocytes and was capable of coupling directly to stimulation of the myocyte contractility.14 In our previous study, the adenosine A1 receptor pathway was blocked by prior treatment of the ventricular myocyte with pertussis toxin to unmask the stimulation of contractility by the various adenosine agonists. The stimulatory effect of adenosine agonists in the primary cultures of fetal chick heart is highly reproducible and can be readily quantified. These cultured heart cells therefore represent a useful cellular model for the study of the cardiac function of adenosine A2 receptor and of A2 receptor subtypes. The purpose of the present study is to investigate whether both the A2a and A2b adenosine receptors are expressed in the heart cell and are capable of stimulating myocyte contractility and cAMP accumulation. A number of experimental approaches were used. First, the extent of positive inotropic responsiveness to adenosine and N-ethyladenosine-5'-uronic acid (NECA), both capable of activating the A2a and the A2b subtypes, was compared with that of the positive inotropic response elicited by the A2a-selective agonist 2-[p-(4-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Second, the effects of antagonists selective at the A2a receptor [8-(3-chlorestyryl)caffeine (CSC)] and at the A2b receptor (1,3-diethyl-8-phenylxanthine [DPX]) and alloxazine on the agonist-stimulated increase in contractile amplitude were determined. Finally, the A2a receptor pathway was selectively desensitized by pretreatment of the myocyte cultures with the A2a-selective agonist CGS21680, and contractile effects of adenosine agonists in control and desensitized myocytes were obtained. Since both A2a and A2b receptors have been demonstrated to couple to stimulation of cAMP accumulation,15 16 23 parallel experiments were also carried out to determine the effects of the same adenosine agonists and antagonists on the cAMP level, which allowed comparison with data obtained in the contractility study.
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Materials and Methods |
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Materials
The adenosine analogues NECA, CGS21680, and DPX were from Research Biochemicals Inc. Adenosine was obtained from Sigma Chemical Co. Alloxazine was purchased from Aldrich Chemicals. Pertussis toxin was from List Biological Laboratories, Inc. Adenosine deaminase was obtained from Boehringer Mannheim Corp. CSC was a generous gift from Dr Kenneth Jacobson (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health). Embryonic chick eggs were from Spafas Inc. The cAMP radioimmunoassay kit was obtained from Amersham.
Methods
Tissue Preparation
Ventricular myocytes were cultured from chick embryos 14 days in
ovo according to a previously described procedure.14
Briefly, isolated ventricular myocytes from chick embryos 14 days in
ovo were prepared in calcium- and magnesium-free Hanks' balanced salt
solution (HBSS) containing 0.025% trypsin (GIBCO). After
neutralization of trypsin with medium containing horse serum and HBSS,
cells were centrifuged and resuspended in culture medium containing 6%
fetal bovine serum, 40% medium 199 (GIBCO), 0.1%
penicillin/streptomycin, and a salt solution. The final concentrations
in the culture medium were (mmol/L) Na+ 142, K+
3.3, Mg2+ 0.7, Ca2+ 1.4, Cl- 130,
HCO3 16.4, and glucose 5.5. Cells were plated at a density
of 400 000 cells per milliliter and cultivated in a humidified 5%
CO2/95% air mix at 37°C. Cells became confluent on day 3
in culture, and contractility measurement was carried out on that day.
Determination of Contractile Amplitude
Measurement of contractile amplitude in cultured ventricular
cells was carried out according to a previously described
method.14 24 Briefly, ventricular myocytes became adhered
to coverslips at the bottom of the dish during culturing. Monolayers of
myocytes exhibit spontaneous rhythmic beating by day 3 of culturing.
Coverslips containing beating cells were placed in a perfusion chamber
situated on the stage of an inverted phase-contrast microscope (Nikon)
with an inlet and an outlet that allowed infusion and removal of medium
containing the various adenosine analogues. The contractile amplitude
of the cultured cell was determined by an opticovideo motion detection
system with a video motion analyzer (Colorado Video), as previously
described. The perfusion medium contained the various adenosine
analogues indicated as well as the following components (mmol/L): HEPES
4 (pH 7.4), NaCl 137, KCl 3.6, MgCl2 0.5, CaCl2
0.6, and glucose 5.5, along with horse serum at 6%. Measurement of
contractile amplitude was carried out on only one cell per coverslip,
and each culture dish contained five coverslips. After achieving a
steady state of beating in medium without adenosine analogues, the
medium was switched to that containing the indicated adenosine
drugs. Both the basal contraction amplitude and the amplitude measured
during adenosine analogue exposure were determined. The stimulatory
effect of the various adenosine analogues on the contractile state was
predominantly on the amplitude of contraction.14 The basal
rate of contraction was 105±16 (mean±SD, n=311). There was no
significant consistent effect of any of the analogues on the
spontaneous rate of contraction.
Prior Treatment of Cultured Myocytes
Cultured ventricular cells were treated with pertussis toxin (5
ng/mL of culture medium) for 24 hours to uncouple the adenosine
A1 receptor from its effector(s) as previously
described.14 This dose and duration of pertussis toxin
treatment were sufficient to cause complete ADP-ribosylation of the
inhibitory G protein by the endogenous NAD+, because
treatment with the same dose of pertussis toxin for only 12 hours
resulted in nearly complete ADP-ribosylation of Gi by the
endogenous NAD+14 and because the ability of
A1 agonist to inhibit the isoproterenol-stimulated increase
in myocyte contractility in these treated cells was completely
abolished (data not shown). Blocking of the A1 receptor
pathway allowed expression and quantification of the adenosine
A2 receptor–mediated increase in myocyte contractile
amplitude or cAMP level. Cultures were also treated with adenosine
deaminase (2 U/mL) for 24 hours to keep the endogenous adenosine at a
minimal level.14 25
Measurement of cAMP Level
Cultured ventricular cells were treated with pertussis toxin and
adenosine deaminase as described above. On day 3 of culturing, the
media were replaced with culture media lacking fetal bovine serum, and
cells were incubated with the indicated agonist(s) and antagonist(s) in
the presence of phosphodiesterase inhibitor rolipram (30 µmol/L).
cAMP was then extracted with the addition of 1/10 vol of 1N HCl to the
media, followed by boiling for 10 minutes. The extracted cAMP was
assayed according to a previously described radioimmunoassay method
(Amersham).17 Recovery of exogenously added cAMP was
98±1.8% (n=15). The effect of agonist on cAMP accumulation was linear
for >10 minutes, and cells were exposed to the indicated adenosine
analogues for 10 minutes before extraction of cAMP. As a positive
control, the cAMP level was measured in the presence of isoproterenol,
which typically caused a 10±1.5-fold (mean±SEM, n=4) increase in
cAMP, with the maximal response occurring at 0.1±0.01 µmol/L (n=4).
Analysis of Data
Stimulation of myocyte contractility was quantified by
expressing the data as percent increase above the basal level of
amplitude; stimulation of cAMP accumulation was determined by
expressing the data as increase in femtomoles of cAMP per milligram
protein. To analyze the change in contractile amplitude or cAMP level
during exposure to both agonists and antagonists, the increase in
contraction amplitude or cAMP determined in the presence of the agonist
and the antagonist was normalized to the increase obtained during
exposure to the agonist alone as percent maximum. One-way ANOVA and
unpaired t test were carried out on data expressed in
percentages (untransformed). The dose-response curve
represented the increase in contractile amplitude in
response to sequentially higher concentrations of the indicated
agonist. In studies of the effects of adenosine analogues on the cAMP
level, the EC50 values and Hill coefficient were obtained
from dose-response curves by computer-aided nonlinear regression
analysis17 (GRAPHPAD software). The
equations used for one-site and two-site analyses were
Y=(BmaxX)/(Kd+X) and
Y=(Bmax1X)/(Kd1+X)+(Bmax2X)/(Kd2+X),
respectively, where Y is the level of cAMP and X is the concentration
of the adenosine receptor agonist. The goodness of fit was determined
by the R2 values, by comparing the sum of
squares when fitting with one-site versus two-site equations, and by F
test.
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Results |
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Differential Inotropic Responsiveness to Adenosine Receptor Agonists
To determine whether both adenosine A2a and A2b receptors are capable of mediating a positive inotropic response, the adenosine A1 receptor pathway was inactivated by pretreating the ventricular myocyte cultures with pertussis toxin (5 ng/mL for 24 hours). The extent of positive inotropic responsiveness caused by NECA or adenosine, which was capable of activating both the A2a and A2b receptors, was compared with that of the positive inotropic responsiveness induced by the A2a-selective agonist CGS21680. The extent of positive inotropic response was quantified by measuring the increase in contractile amplitude above the basal level in the presence of each agonist. All three adenosine agonists caused a dose-dependent increase in contractile amplitude (Fig 1
). However, the maximal
increase in contractile amplitude elicited by adenosine (27±1.6%
[mean±SEM, n=29]) or NECA (27.6±3% [n=29]) was significantly
greater than the increase stimulated by CGS21680 (16.5±1.2% [n=47])
(one-way ANOVA [F=10.2, P=.0001], followed by unpaired
t test between the groups [P<.01 for the
t values]). On the other hand, the maximal positive
inotropic responses caused by adenosine and NECA were similar to each
other (Figs 1 and 2) (t=0.2,
P=.84). The increase in contractility caused by NECA or
adenosine was also greater than the increase elicited by CGS21680 at 10
µmol/L of the agonists (ANOVA, followed by group comparison [F=3.66,
P=.028]). Such differential positive inotropic
responsiveness indicated that adenosine or NECA stimulates cardiac
myocyte contractility via a pathway(s) that is different from that
activated by CGS21680 and raised the possibility that both
A2a and A2b receptors may be involved in
mediating the positive inotropic response to adenosine and NECA.
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Differential Effects of the A2a-Selective Antagonist
CSC on Positive Inotropic Response Stimulated by CGS21680, NECA, and
Adenosine
CSC was recently demonstrated to be a highly selective adenosine
A2a receptor antagonist both in vitro and in
vivo.26 27 The ability of CSC to inhibit the increase in
contractility caused by CGS21680, NECA, and adenosine was compared (Fig 3). Although the selectivity of CSC for A2a
versus A2b receptors is not known, the inhibitory effect of
CSC on the CGS21680-stimulated increase in contractile amplitude
differed from that of CSC on the adenosine- or NECA-induced
increase in contractility. CSC, at 1µmol/L, inhibited nearly
completely the increase in myocyte contractile amplitude caused by
CGS21680 (Figs 3, top, and 4), indicating that CSC was
capable of antagonizing the A2a receptor–mediated increase
in myocyte contractility in these fetal chick ventricular myocytes. On
the other hand, CSC was able to block only part of the increase in
contractility stimulated by NECA or adenosine (Figs 3, middle and
bottom, and 4). Data were expressed as percent maximal inotropic
response to each agonist versus CSC concentrations. The extent of
CSC-induced inhibition was inversely related to the percent maximum,
such that with increasing CSC inhibition, there was a progressive
decrease in the percent maximum. At either 0.1 or 1 µmol/L CSC, the
percent maximal NECA- or adenosine-induced stimulation of
contractility was significantly greater than the percent maximal
stimulation elicited by CGS21680 (one-way ANOVA [F=18.8 and 59.1 for
0.1 and 1 µmol/L CSC, respectively, P<.001]; ANOVA was
then followed by t test [P<.001]). These data
indicated that the extent of CSC-mediated inhibition of NECA- or
adenosine-induced positive inotropic response was less than the
CSC inhibition of CGS21680-stimulated contractile response. On the
other hand, values for the inhibition of NECA- or
adenosine-stimulated contractility by CSC were similar to each
other (P>.05). Thus, CSC blocked the portion of the
positive inotropic effect of adenosine or NECA that was mediated via
A2a receptors.
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Effect of Selectively Desensitizing the Adenosine A2a
Receptor on the Positive Inotropic Response to CGS21680 and NECA
To provide further evidence for the notion that the partial
blocking of NECA- or adenosine-stimulated increase in
contractility by CSC was due to its antagonism of the effect of NECA at
A2b receptors, ventricular myocyte cultures were pretreated
with 30 nmol/L CGS21680 for 24 hours to selectively desensitize the
A2a receptor pathway. Because CGS21680 is selective at the
A2a receptor and because, at 30 nmol/L, the A2
receptor that would be activated is primarily the A2a
subtype, it is likely that such pretreatment would desensitize
selectively the A2a receptor. In myocytes pretreated with
CGS21680 and subsequently washed free of the agonist, 30 µmol/L of
CGS21680 was not able to elicit a significant positive inotropic
response (from percent increase in contractile amplitude of 17.3±2%
[mean±SEM, n=36] to percent increase of 2.9±1.4% [n=29]) (Figs 5 and 6). On the other hand, prior
treatment of the ventricular cultures with CGS21680 abolished only part
of the NECA (30 µmol/L)–induced positive inotropic response (Figs 5 and 6). Nearly 50% of the NECA-induced increase in contractile
amplitude remained after selective desensitization of the
A2a receptor (from a percent increase in contractile
amplitude of 25±1.6% [n=42] to a percent increase of 12.1±1.5%
[n=33]). Such data indicate that the stimulatory effect of NECA on
myocyte contractility was mediated by not just the A2a
subtype but also by another stimulatory adenosine receptor, likely the
A2b receptor.
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Effects of the A2b-Selective Antagonists DPX and
Alloxazine on the Positive Inotropic Response to CGS21680, NECA, and
Adenosine
The finding that only part of the positive inotropic effect of
adenosine and NECA was blocked by CSC or by the selective
desensitization of the A2a receptor pathway indicated that
another stimulatory adenosine receptor is also involved in mediating
the positive inotropic effect of these agonists. To demonstrate that
this other receptor subtype is the A2b receptor, the effect
of A2b-selective antagonist DPX on the positive inotropic
response to adenosine and NECA was compared with that of DPX on
the positive inotropic response to CGS21680. Although DPX is primarily
an A1-selective antagonist, in cells in which the
A1 receptor pathway has been inactivated by prior treatment
with pertussis toxin, DPX could function as an A2b
receptor–selective antagonist. Previous studies demonstrated that DPX
was 
20-fold more selective for the A2b receptor than it
was for the A2a receptor.28 DPX, at 10
µmol/L, caused only a modest inhibition of the CGS21680-stimulated
increase in contractile amplitude (percent maximum, 75.6±3.3%
[meant±SEM, n=13]) (Figs 7, top, and
8). On the other hand, DPX caused a
pronounced inhibition of the NECA-stimulated (percent maximum,
44.8±5% [n=24]) and of the adenosine-stimulated (percent
maximum, 40.1±6.3% [n=12]) increase in contractile amplitude. The
percent maximal NECA- or adenosine-elicited stimulation
of myocyte contractility was significantly less than the percent
maximal CGS21680 stimulation at either 1 µmol/L DPX (one-way ANOVA
[F=10.8, P=.0001], followed by t test
comparison between groups) or 10 µmol/L DPX (ANOVA [F=10.42,
P=.0002], followed by t test comparison between
groups). Thus, DPX was more efficacious in inhibiting the
adenosine- or NECA-induced stimulation of contractility than it
was in blocking the CGS21680 response. Finally, DPX was able to abolish
the part of the NECA-induced increase in contractile amplitude that
remained after blocking the effect of NECA with CSC (Fig 9). The inhibition of NECA- and
adenosine-stimulated increase in contractility at the highest
concentration of DPX was partial, consistent with the notion that part
of the positive inotropic effect of adenosine or NECA is mediated by
the A2b receptor.
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Similar data were obtained for the effects of alloxazine on the
CGS21680- versus NECA-stimulated increase in myocyte contractility (Fig 10, top and middle). Alloxazine showed some selectivity
at A2b compared with A2a
receptors.29 The percent maximal CGS21680-stimulated
contractile response (88±2.5% [mean±SEM, n=18]) was greater than
the percent maximal NECA response (60.5±6.4% [n=18]) in the
presence of 1 µmol/L alloxazine (t=4.47,
P<.0001); similarly, at 10 µmol/L alloxazine, the percent
maximal CGS21680 response (80±4% [n=18]) was greater than the
percent maximal NECA response (50±6.5% [n=20]) (t=4.2,
P<.001). These data indicated that alloxazine was able to
cause a greater inhibition of the NECA- than of the CGS21680-elicited
positive inotropic response.
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Differential Inhibitory Effects of CSC on CGS21680- Versus
NECA-Induced Stimulation of cAMP Accumulation
The A2a-selective antagonist CSC blocked only part of
the NECA-mediated increase in cAMP accumulation, whereas it abolished
most of the CGS21680-induced increase in cAMP level (Fig 11). At each concentration, CSC caused a significantly
greater inhibition of the CGS21680-stimulated than of the
NECA-stimulated increase in cAMP level. Data were expressed as percent
maximal stimulation of cAMP accumulation by each agonist in the
presence of increasing CSC concentrations. At each CSC concentration,
the percent maximal NECA-stimulated increase in cAMP was significantly
greater than the percent maximal stimulation elicited by CGS21680
(P<.05, t test). Since the extent of CSC-induced
inhibition was inversely related to the percent maximum, these data
indicated that the extent of CSC-mediated inhibition of NECA-induced
increase in cAMP was less than the CSC inhibition of
CGS21680-stimulated increase in cAMP. Such data confirm that CSC is an
A2a-selective antagonist in these cultured myocytes and
that another stimulatory adenosine receptor is present and can
mediate an increase in cAMP accumulation.
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Effects of DPX on CGS21680- Versus NECA-Induced Stimulation of cAMP
Accumulation
To demonstrate the involvement of A2b receptors
in mediating the NECA-induced increase in cAMP accumulation, the
effects of the A2b receptor–selective antagonist DPX on
the cAMP response to NECA was compared with that of DPX on the CGS21680
response. DPX was more efficacious in inhibiting the NECA-stimulated
than the CGS21680-stimulated increase in cAMP accumulation. The percent
maximal NECA (10 µmol/L)–stimulated increase in cAMP accumulation
was significantly less than the percent maximal CGS21680 (3 µmol/L)
stimulation at 1 µmol/L DPX (percent maximal NECA response, 17±3%
[mean±SEM]; percent maximal CGS21680 response, 77±8% [n=5]) or
10 µmol/L of DPX (percent NECA response, 5±2.4%; percent CGS21680
response, 49±4% [n=5]) (t test, P<.01). Such
a differential response of the nonselective versus the
A2a-selective agonist to the A2b receptor
blocker is similar to the results obtained in parallel studies carried
out on the contractility response (Figs 7 and 8). Finally, the addition
of DPX (10 µmol/L) to CSC (1 µmol/L) resulted in 95±2% (n=3)
inhibition of the NECA-stimulated increase in cAMP level (control,
27.8±0.85 pmol cAMP/mg; NECA alone, 216±18.5; and NECA plus CSC and
DPX, 36.4±1.05 [mean±SEM, n=3]).
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Differential Stimulatory Effects of Adenosine Receptor Agonists on
the cAMP Level
If NECA was able to stimulate cAMP accumulation via activation of
the A2a receptor at low concentrations and via the
A2b receptor at higher concentrations, stimulation of cAMP
accumulation by NECA in the presence of CSC should reflect activation
of only the A2b receptor and should exhibit a dose-response
curve that is different from that obtained in the absence of CSC. NECA
caused a biphasic dose-dependent increase (an average 11.2±2.7-fold
stimulation) in the cAMP level with a Hill coefficient of 0.62±0.07
(n=5); however, in the presence of 1 µmol/L CSC, the NECA
dose-response curve became steep and monophasic with a Hill coefficient
of 1.2±0.25 (n=5) (Fig 12). Although the dose-response
curve obtained in the presence of NECA alone was clearly biphasic,
nonlinear regression analysis could not yield the EC50
values for high- and low-affinity sites, because the high-affinity
sites appeared to account for only 10% of the total sites; ie, the
curve exhibited a very low plateau. Thus, the response to NECA over the
range of 0.003 to 0.3 µmol/L was first fit to a one-site model.
Although the number of concentrations of NECA over the range of 0.003
to 0.3 µmol/L were limited, the monophasic nature of the curve with a
Hill coefficient close to 1.0 (0.87±0.05) provides evidence for a
one-site model fit of the data points. Such analysis yielded the
Bmax and EC50 values of the high-affinity NECA
sites (Table). The Bmax and EC50
values thus derived were then entered as constants for the
high-affinity sites in the two-site analysis of the entire NECA
dose-response curve, which yielded Bmax and
EC50 values for the low-affinity sites. The low-affinity
Bmax and EC50 values derived from such two-site
analyses were similar to those derived from the one-site model of the
NECA dose-response curve that was obtained in the presence of CSC
(Table). On the other hand, the A2a-selective agonist
CGS21680 caused a monophasic stimulation of cAMP accumulation (Hill
coefficient, 0.88±0.1; average increase, 0.95±0.2-fold [n=5]) (Fig 12), which was best fit by a one-site model (no improvement, F test).
The EC50 value for the high-affinity NECA sites was similar
to that for CGS21680 (Table), consistent with the notion that the
high-affinity NECA sites represent the A2a
receptors.
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Discussion |
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Heterogeneity of cell-surface receptors is usually due to the existence of multiple receptor subtypes. Subtypes of the same class of receptor exhibit either tissue specificity or function specificity. Although multiple subtypes of the same class of receptor have been shown to coexist and mediate different functions on the heart cell,1 2 3 4 5 6 7 8 9 whether two separate receptor subtypes with different affinity can couple to the same functional responses in the same cardiac cells is not known. Demonstration of such coexistence would suggest a novel feature of cell regulation, ie, a dual regulatory role of the receptor agonist and its high-affinity and low-affinity receptor subtypes. In studies of cardiac action of adenosine and function of adenosine receptor subtypes using cultured fetal chick heart cells as a model, high-affinity (A2a) and low-affinity (A2b) adenosine receptor subtypes were found to coexist but were coupled to the same functional responses, mediating stimulation of myocyte contractility and cAMP accumulation.
Adenosine exerts pronounced biological effects in the heart.10 11 12 13 The inhibitory effects of adenosine on the sinus and atrioventricular node functions and on the ß-adrenergic–stimulated increase in myocyte contractility are mediated by the A1 subtype.10 11 12 13 Data have accumulated to demonstrate both the existence and the absence of a functional adenosine A2 receptor on the myocardium.14 18 19 20 21 22 30 31 32 33 The reasons for the conflicting data on the existence of a functional A2 subtype on the cardiac myocyte are not entirely clear. A number of explanations are possible. First, the use of acutely dispersed cardiac myocytes as a model to study the expression of a functional A2 receptor is complicated by the variable presence of proteases in different batches of the collagenase and trypsin used to dissociate the myocytes. Since the A2 receptor that was protease-digested exhibited reduced association with the stimulatory G protein,34 the use of different batches of such enzymes may cause a differential proteolysis of the cell-surface A2 receptor, resulting in uncoupling or degradation of the receptor. Second, adenosine released during cell isolation may cause desensitization or even downregulation of the A2 receptor. Third, since activation of the A1 receptor inhibits the stimulatory effect mediated by the A2 receptor, incomplete inactivation of the A1 receptor pathway may mask the stimulatory effect produced by A2 receptor activation. Fourth, the presence of fibroblasts and endothelial cells, which express the A2 receptor coupled to the stimulation of adenylyl cyclase, in slices or membranes of heart tissues may also account for the stimulatory effect of adenosine agonists in such preparations. Our previous study demonstrated that the adenosine A2 receptor was present on ventricular but not atrial myocytes cultured from 14-day chick embryos and was coupled directly to stimulation of the myocyte contractility. The potential advantages of these cultured myocytes include the facts that the myocytes and their receptors could recover from the cell isolation procedure during the 48 to 72 hours of cultivation, that adenosine released into the culture medium can be kept to a minimum with the addition of adenosine deaminase, and that prolonged treatment of the myocyte cultures with pertussis toxin resulted in complete ADP-ribosylation of Gi from the endogenous NAD+ and thus in the uncoupling of the A1 receptor from its effectors.14
The objective of the present study, using ventricular cells cultured from 14-day chick embryos, was to test whether both the A2a and the A2b receptors mediate the stimulatory effect of adenosine on the myocyte contractility and cAMP accumulation. Measurement of cAMP accumulation provided another functional response that is mediated by the adenosine receptor.10 11 12 13 14 15 16 Since cAMP measurement was carried out under similar conditions in the same cultured myocytes, determination of the cAMP response would facilitate direct correlation with results obtained in the contractility study. Further, since A2b receptors could only be demonstrated to stimulate cAMP accumulation in intact brain slices but not in membranes,15 measurement of cAMP in the intact myocytes will allow quantification of not only an A2a receptor–mediated response but also an A2b receptor–mediated response. A number of lines of evidence were provided to support the notion that both A2 receptor subtypes coexist and are coupled to stimulation of myocyte contractility and cAMP accumulation. First, NECA and adenosine, both capable of activating A2a and A2b subtypes, caused a greater increase in myocyte contractility than did the A2a-selective agonist CGS21680. The significantly greater stimulation of myocyte contractility by NECA or adenosine compared with that caused by CGS21680 occurred at >1 µmol/L of the agonists, consistent with the notion that at the lower concentrations, all three agonists activated primarily the A2a receptor, whereas at concentrations >1 µmol/L, NECA and adenosine stimulated the A2b receptor and hence produced a greater positive inotropic response. NECA also caused a biphasic increase in cAMP accumulation, whereas the A2a receptor–selective agonist CGS21680–elicited response was monophasic, consistent with the notion that NECA interacted with high-affinity (A2a) and low-affinity (A2b) sites. The NECA-induced cAMP response became monophasic in the presence of the A2a receptor–selective antagonist CSC. Second, the A2a-selective antagonist CSC, although capable of inhibiting nearly completely the CGS21680-elicited positive inotropic and cAMP responses, could only block part of the responses elicited by NECA or adenosine. CSC has been shown to be an A2a-selective antagonist in radioligand binding assays in the rat brain and in antagonism of the A1 agonist effect on cAMP accumulation in rat adipocytes versus that of the A2a agonist effect in rat pheochromocytoma cells.26 27 In the present study, CSC caused a much more pronounced inhibition of the CGS21680-stimulated than of the NECA-induced increase in contractile amplitude and in cAMP level. Such data are consistent with the notion that CSC is a more selective antagonist at the A2a than at the A2b receptors in these cultured myocytes. Third, selective desensitization of the A2a receptor by prior treatment of the myocytes with low concentrations of CGS21680 resulted in desensitization of the CGS21680-induced stimulation of contractility and only a partial disappearance of the positive inotropic effect of NECA. It is possible that 30 nmol/L CGS21680 treatment for 24 hours also desensitized some of the A2b receptor and that 1 µmol/L CSC also blocked some of the A2b receptors. Nevertheless, the simplest and most consistent explanation for these data is that blocking with CSC and pretreatment with 30 nmol/L CGS21680 resulted in a selective antagonism at and desensitization of, respectively, the A2a receptor. Finally, the A2b-selective antagonists DPX or alloxazine caused a more pronounced inhibition of the increase in contractility or cAMP accumulation elicited by NECA than the inhibition caused by CGS21680. Previous studies indicated that DPX28 and alloxazine29 are relatively more selective at the A2b than at the A2a receptor. Both antagonists caused greater inhibition of the NECA-induced than of the CGS21680-induced positive inotropic response. The differential inhibitory effect of DPX on NECA- versus CGS21680-induced contractile response paralleled that of the DPX on NECA- versus CGS21680-stimulated cAMP response. Although DPX is not very selective at the A2b compared with the A2a receptors, such differential inhibitory effects suggest that the A2b receptor mediates part of the contractile and the cAMP responses. Finally, the finding that DPX could antagonize part of the NECA-stimulated increase in contractility or in cAMP that could not be blocked by CSC provides additional evidence that functional A2b receptors are expressed on these heart cells.
A number of considerations are needed in interpreting the present data. After blocking with CSC or desensitizing with CGS21680, the magnitude of the increase in contractile amplitude is small. Thus , it was not feasible to obtain a dose-response relation for the A2b receptor–mediated stimulation of contractility nor was it possible to compare the affinity of the A2a receptor with that of the A2b receptor for adenosine or NECA in the contractility study. In the absence of full A2a or A2b receptor–mediated dose-response curves, where the contractile effect mediated by the other subtype can be completely eliminated, the definitive and exact contribution of each receptor subtype to the positive inotropic effect of adenosine remains unknown. However, because the magnitude of stimulation of cAMP by NECA and CGS21680 could be easily quantified, the affinity of both subtypes could be determined by nonlinear regression analysis of CGS21680 and NECA concentration response curves, yielding EC50 values. Similarity between EC50 obtained in the CGS21680 curve and EC50 of the high-affinity sites determined in the NECA dose-response curve suggests that they represent estimates of the A2a receptor affinity. Similarity between EC50 of the low-affinity site determined in the NECA dose-response curve and EC50 for NECA determined in the presence of CSC indicates that such EC50 values represent the estimate of the A2b receptor affinity. Whether such estimates (ie, EC50 values) of affinity for the two receptor subtypes represent the affinity constant of the two receptor subtypes is not known, because the increase in cAMP level that was used to determine the EC50 values was influenced not just by the affinity constant of the receptor for the agonist but also by the efficiency of receptor–G protein–adenylyl cyclase coupling. Nevertheless, such estimates of affinity for A2a and A2b receptors agree with those obtained in studies of cAMP response to adenosine agonists in striatum, PC12 cells, and fibroblasts17 23 29 and suggest that A2a and A2b receptors on these cultured myocytes share similar pharmacological features, such as the A2a and A2b receptors, in other tissues. Finally, CGS21680 (acting through A2a receptors) appeared to cause a larger increase in contractility relative to its ability in stimulating cAMP accumulation when compared with the ability of NECA, in the presence of CSC, to stimulate contractility versus cAMP accumulation (acting through A2b receptors). Such a finding raises the possibility that the mechanism(s) underlying A2a receptor– and A2b receptor–mediated augmentation of myocyte contractility may be different, which deserves further study.
Overall, the present study provides the first direct evidence for the coexistence of two receptor subtypes, the adenosine A2a and A2b receptors, which have greatly different affinities but are coupled to the same functional responses in the heart cells. Further, the present study demonstrated for the first time that a functional A2b receptor is expressed and is capable of mediating augmentation of cardiac myocyte contractility. Whether both A2a and A2b receptors coexist and are functional in the adult heart cell is not known. The present data suggest a novel feature of fetal heart cell regulation whereby the agonist adenosine and its high-affinity (A2a) and low-affinity (A2b) receptors play dual regulatory roles. Under physiological conditions in which a low level of adenosine is released in the heart, the high-affinity A2a receptor may be an important modulator, whereas under pathophysiological conditions, such as cardiac ischemia, in which a large amount of adenosine is released, the low-affinity A2b receptor can assume functional significance.
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Acknowledgments |
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This study was carried out during the tenure of an Established Investigatorship of the American Heart Association and was supported by grant RO1-HL-48225 from the National Institutes of Health (Dr Liang). The authors wish to thank Dr Kenneth A. Jacobson for supplying 8-(3-chlorostyryl)caffeine and for useful discussions on the relative selectivity of alloxazine as an A2b-selective antagonist and Dr Joel Linden for useful discussions on the properties of 1,3-diethyl-8-phenylxanthine.
Received April 6, 1994; accepted October 5, 1994.
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