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Vol. 285, Issue 1, 186-192, April 1998
Department of Pharmacology, MCP 
Hahnemann School of Medicine,
Allegheny University of the Health Sciences, Philadelphia, Pennsylvania
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Abstract |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Previously published reports from this laboratory have shown that the
antiadrenergic effect of adenosine A1 agonists declines with age in the rat heart [Gao et al. (1997)
J
Mol Cell Cardiol 29:593-602] and that this decline
may be caused by a decrease in coupling between adenosine
A1 receptors (AdoA1R) and guanine nucleotide-binding proteins [Cai et al. (1997)
Circ Res 81:1065-1071]. Dietary restriction
(DR; 60% calories of ad libitum) has been shown to
attenuate age-related changes in cellular signal transduction pathways.
Therefore, the present study investigated whether DR altered the
age-related changes in AdoA1R-mediated function in senescent rat hearts. Ventricular membranes were isolated from the
hearts of ad libitum (AL) fed and DR male F344 rats that
were 6, 12 and 24 months of age. In AL rats, there was an age-related decline in isoproterenol (ISO)-stimulated adenylyl cyclase when compared with the 6-month-old rats. The decline in ISO-stimulated cyclase was attenuated in DR animals. In AL rats, inhibition of ISO-stimulated adenylyl cyclase by the AdoA1R agonist,
N6-p-sulfophenyladenosine (SPA) decreased
with age. In DR rats, the age-related decline in inhibition was
attenuated. Previous results from this laboratory indicated that in AL
fed rats, there was an age-related decrease in the percentage of
high-affinity binding sites for SPA, from 55% at 6 months to 23% at
24 months. Diet restriction attenuated this age-related shift in
high-affinity binding sites so that the percentage of high-affinity
sites at 24 months was 42%. Our results suggest that DR maintains
AdoA1R function by preventing a loss of high-affinity
AdoA1R sites.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Adenosine
is a byproduct of ATP degradation which is released from myocytes under
ischemic and hypoxic conditions (Sparks and Bardenhauer, 1986). The
antiadrenergic action of adenosine reduces oxygen demand when oxygen
supply is limited (Huang and Drummond, 1976; Belardinelli and Isenberg,
1983; Achterberg et al., 1985; Romano et al.,
1991). Postsynaptic inhibition of myocyte responses to beta
adrenoceptor stimulation are mediated through the
AdoA1R coupled to the inhibitory guanine
nucleotide-binding protein Gi (Evans and Schenden
1982; Linden et al., 1985; Martens et al., 1987;
Henrich et al., 1987).
Studies from our group have shown that
AdoA1R-mediated antiadrenergic effects decline
with age in the rat heart (Gao et al., 1997). The specific
AdoA1R agonists,
N6-cyclopentyladenosine (Bruns et al.,
1986) and SPA (Jacobsen et al., 1992); inhibit ISO- and
forskolin-stimulated adenylyl cyclase activity in cardiac membranes in
6-month-old but not in 24-month-old male F344 rats (Gao et
al., 1997). We have also shown that the age-related decrease in
AdoA1R function in the ventricle may be caused by
a decrease in AdoA1R/G-protein coupling (Cai
et al., 1997). Thus the protective actions of adenosine may
be diminished in the elderly heart.
Restriction of caloric intake to 60% - 70% of ad libitum
intake postpones the occurrence of pathology, extends life-span in the
rat (Snyder et al., 1990; Masoro, 1993) and has been shown to attenuate the age-related changes in several physiological processes
(Yu, 1994). These include age-related changes in the following
parameters of signal transduction pathways: beta adrenergic receptors in the liver (Dax et al., 1989), beta
adrenergic stimulation of adenylyl cyclase in the liver (Katz, 1988)
and lung (Scarpace and Yu, 1987) and alpha adrenergic
receptors in the aorta (Gurdal et al., 1995) and parotid
gland (Chen et al., 1997). The present study further
characterizes the age-related changes in beta adrenergic receptor- and AdoA1R-mediated function in
senescent rat hearts by investigating these receptor systems in DR
rats.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Reagents.
Adenosine 5'-triphosphate, tetra
(triethylammonium) salt, [
-32P]
(32P-ATP) and 3H-DPCPX were
obtained from NEN (Boston, MA). Adenosine-3'5'-cyclic phosphate
[2,8-3H], ammonium salt
(3H-cAMP) was obtained from American Radiolabeled
Chemicals (St. Louis, MO). SPA and 8-SPT were obtained from Research
Biochemical International (Natick, MA). 5'-Guanylylimidodiphosphate
(Gpp(NH)p), adenosine deaminase and other chemicals as well as
chromatographic alumina WN-3 were purchased from Sigma Chemical Company
(St. Louis, MO). Dowex AG50 WX4 cation exchange resin (200-400 mesh)
was purchased from Bio-Rad (Melville, NY).
Animals. These studies have been carried out according to the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Male 6-, 12-, and 24-month-old Fisher 344 rats were reared and maintained at the National Center for Toxicological Research (NCTR) animal colony. These rats were fed either AL or restricted to 60% of the food intake of the ad libitum fed rats (DR) from the time of weaning. They were shipped approximately 1 month before use. In our institution, rats were housed in a barrier facility, in standard filter topped cages, one rat per cage. Room temperature was set at 21 ± 1°C; the light/dark cycle was 12 h; humidity was controlled at 40 to 65%. The animals were fed a pasteurized rodent diet and autoclaved water adjusted to pH 3.
Membrane preparation for adenylyl cyclase assay.
Crude
cardiac ventricular membranes were prepared from rat hearts as
described previously (Gao et al., 1997). Rats were
decapitated and the hearts were quickly excised and stored in ice-cold
buffer containing 10% sucrose, 1 mM EGTA and 5 mM Tris-HCl at pH 7.4. Atria were detached and ventricles were minced and homogenized for
10 s with a polytron set at power 6. The suspension was
centrifuged at 1,000 × g for 10 min, and the
supernatant was then centrifuged at 27,000 × g for 20 min. The pellet was washed twice and resuspended in 100 mM Tris-HCl
buffer (pH 7.4). Protein concentration was measured by the method of
Bradford (1976). All the membrane preparations were frozen at 
70°C.
Membrane preparation for adenosine A1
receptor binding assay.
The cardiac membranes used for receptor
binding were prepared according to the method of Lee et al.
(1993) with minor modification. Hearts were washed in ice-cold
calcium-free phosphate-buffered saline containing 1 mM EDTA. The
ventricles were homogenized as above. The suspension was centrifuged at
1,000 × g for 10 min, and the supernatant was then
centrifuged at 49,000 × g for 20 min. The pellet was
washed twice and resuspended in a buffer containing 10 mM HEPES, 0.1 mM
EDTA, 0.1 mM benzamidine (pH 7.4). The membrane was incubated with
adenosine deaminase (2 U/ml) at 25°C for 20 min before being stored
at 70°C.
Adenylyl cyclase assay.
Adenylyl cyclase activity was
assayed according to the method of Salomon (1979). Membranes were
thawed and then preincubated for 10 min at 37°C with adenosine
deaminase (5 U/ml) to eliminate endogenous adenosine. The reaction
mixture was prepared on ice with the final concentration of components
as follows: 100 mM Tris-HCl (pH 7.4), 0.1 mM MgATP, 0.6 mM
MgCl2, 1 mM EGTA, 10 µM GTP, 1 mM cAMP, 50 mM
NaCl, 1 mM dithiothreitol, 10 mM creatine phosphate, creatine
phosphokinase (7 U/ml), adenosine deaminase (5 U/ml),
32P-ATP (1 µCi/assay) and
3H-cAMP (0.02 µCi/assay). ISO, forskolin,
AdoA1R agonists and antagonist were added
according to the different protocols. The reaction mixture, containing
everything but cardiac membranes, was preincubated at 37°C for 5 min.
The reaction was initiated by adding membrane to the reaction mixture.
After 30 min the reaction was terminated by adding stop solution that
contained 2% sodium dodecyl sulfate, 25 mM ATP and 1.3 mM cAMP. The
32P-cAMP was isolated from
32P-ATP by the double-column procedure of Salomon
(1979). Column eluates were collected and counted by liquid
scintillation spectrometry. The recovery of cAMP was consistently 80%
as determined by the recovery of 3H-cAMP. In all
cAMP assays, membrane preparations from a single rat were assayed in
triplicate. The data were expressed as picomoles of cAMP formed per
minute per milligram of protein.
AdoA1R receptor binding assay. AdoA1R number in the cardiac membrane preparations was measured by radioligand binding assay with [3H]DPCPX, an AdoA1R antagonist. The reaction mixture (final volume, 0.2 ml) consisted of the following: 400 µg of membrane protein, 1 mM MgCl2, 0.01% CHAPS, 10 mM Tris (pH 7.4) and [3H]DPCPX (109 Ci/mmol, 0.05-4.0 nM). The reaction mixture was incubated at 25°C for 120 min. The reaction was terminated by adding 4 ml of ice-cold Tris buffer, and rapid filtering through Whatman GF/B filters with a Brandel Cell Harvester, followed by one additional wash with the same buffer. Filters were placed in scintillation liquid and counted by liquid scintillation spectrometry. The dissociation constant (Kd) and maximum binding capacity (Bmax) of [3H]DPCPX binding were determined by Scatchard analysis. The competition experiments were performed in the presence of [3H]DPCPX at a fixed concentration, approximately equal to its Kd, and varying concentrations of SPA with or without Gpp(NH)p. Nonspecific binding was determined by use of 330 µM SPA. In this binding assay, the specific binding was between 65 and 73%.
Statistical analysis. All data are expressed as mean ± S.E.M. Differences within and between groups (diet: AL and DR; age: 6, 12 and 24 months of age) were examined by ANOVA with multigroup comparisons, followed by Dunnett's test. Student's t test for unpaired data was used to analyze differences in radioligand Bmax and Kd. Statistical significance was defined as P < .05 and is indicated in the figures and text. EC50 was calculated with the linear regression program of StatView.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effect of age and DR on ISO-stimulated cAMP production. Isoproterenol caused a concentration-dependent increase in the activity of adenylyl cyclase in all age and diet groups (fig. 1). Table 1 indicates the maximal response to ISO at all ages. In AL rats, there was a significant decline in ISO-stimulated cAMP production between 6 and 12 months (36% reduction) and between 6 and 24 months (53% reduction). In contrast, in DR rats, the age-related decline in ISO-stimulated cAMP production was attenuated. There was only a 14% reduction between 6 and 12 months and a 24% reduction between 6 and 24 months. There were no significant differences in the EC50 for ISO between age and diet groups. Because a concentration of 100 µM caused a maximal stimulation at all ages and diets, this concentration was used to stimulate adenylyl cyclase in all subsequent experiments.
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Effect of age and DR on AdoA1R-mediated
inhibition of ISO-stimulated cAMP production.
Previous work from
this laboratory has demonstrated an age-related decline in the
SPA-mediated inhibition of adenylyl cyclase (Gao et al.,
1997). The effect of DR on the age-related decline in the effect of SPA
(100 µM) is shown in figure 2. Basal
and GTP-stimulated cyclase were not affected by either SPA or DR (data not shown). At 6 months, SPA inhibited ISO-stimulated adenylyl cyclase
by 22% in AL rats and 20% in DR rats. Diet restriction attenuated the
age-related decline in the SPA-mediated inhibition of adenylyl cyclase.
At 12 months, SPA-mediated inhibition of adenylyl cyclase in DR rats
was 14%, whereas the inhibition in AL rats was only 5%. Diet
restriction caused a similar attenuation in the 24-month-old rats.
Thus, DR attenuates the age-related decline in
AdoA1R-mediated inhibition of adenylyl cyclase.
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). This falls within the range of the
Ki of 8-SPT for
AdoA1Rs (Bruns et al., 1986; Shamin
et al., 1989). Thus, the inhibition of adenylyl cyclase is
mediated by the A1 receptor subtype at 24 months
as well as at 6 months.
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Effects of age and DR on AdoA1R-mediated
inhibition of forskolin-stimulated adenylyl cyclase activity.
Forskolin (10 µM) directly stimulates adenylyl cyclase activity,
bypassing the need for beta adrenergic receptor stimulation (Seamon and Daly, 1986). Stimulation with forskolin, therefore, provides some indication of adenylyl cyclase activity. In our experiments, forskolin produced a much larger increase in adenylyl cyclase activity than ISO at all ages, which indicates high residual adenylyl cyclase activity even in senescent rat hearts. There was a
significant age-related decline in forskolin-stimulated cAMP production
between 6, 12 and 24 months in AL rats that was attenuated in DR rats
(fig. 4). This suggests some decrease in adenylyl cyclase activity with age that is attenuated by DR.
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Effect of age and DR on AdoA1R density and G-protein coupling. To determine whether DR altered AdoA1R density, receptor number was determined in cardiac membranes from 6- and 24-month-old AL and DR rats by measuring saturation binding of the AdoA1R antagonist [3H]DPCPX (fig. 6). Scatchard analyses of the binding data revealed a linear curve for each age group, reflecting single affinity binding sites for the AdoA1R antagonist. Neither the Bmax nor the Kd changed with age in the AL and DR groups (table 2). Thus, the attenuation of the age-related decline in the effect of SPA is not caused by an increase in receptor number.
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). Analysis of the displacement curve obtained from
similar experiments done with membranes from DR rats also showed two
affinity binding sites at both ages (fig.
7). DR attenuated the decrease in
high-affinity binding sites. Thus, at 24 months the percentage of
high-affinity binding sites is 43% rather than 24% (table
3). The nonhydrolyzable GTP analog,
Gpp(NH)p, shifted the agonist displacement curve for AdoA1R binding to the right in both 6- and
24-month-old DR rats to yield a single low-affinity binding site for
the receptor. This indicates that all the sites can be uncoupled from
G-proteins by adding excess GTP analog.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The present studies confirm our previous observations that
AdoA1R-mediated function decreases with age in
the rat heart (Gao et al., 1997) and that decreased
AdoA1R function is not caused by changes in
AdoA1R number. New to this article is the
observation that DR attenuates the age-related decline in
AdoA1R-mediated function, as well as the decrease
in AdoA1R coupling to G-protein. These studies
provide additional evidence that the protective, antiadrenergic
function of the AdoA1R is diminished in the aged heart.
The age-related decline in AdoA1R function may be
caused by changes in the receptor number with age. However, our studies indicate that there is no change in receptor number with age in either
AL or DR rats. There are reports of increased
AdoA1R density in the senescent heart (Montamat
et al., 1996; Romano and Dobson, 1996). We cannot account
for these differences except for fact that the reported
Kd for [3H]DPCPX in
the Romano and Dobson (1996) study was 2 nM, which is much higher than
the one reported in these studies. Furthermore, in those studies the
Kd increased with age, whereas in our study there was no change with age. The study of Montamat et al.
(1996) was done in atria rather than ventricle. Perhaps different
methods of membrane preparation could account for the differences.
An age-related change in the number of G
i
guanine nucleotide regulatory proteins could also account for the
decline in AdoA1R function in old male rat
hearts. The alpha subunit of Gi is
present in three isoforms (Gi1,
Gi2,
Gi3) which have been identified by
molecular cloning (Birnbaumer et al., 1985; Itoh et
al., 1986; Jones and Reed, 1987). All these isoforms have been
shown to inhibit adenylyl cyclase activity (Kobayashi et
al., 1990). Studies from our laboratory have demonstrated that AdoA1R is coupled to
Gi3 and Go
(Cai et al., 1997). Consistent with previous studies (Shu
and Scarpace 1994; Johnson et al., 1995), we have found no
change in Gi levels or the distribution
among the isoforms in the hearts of F344 rats between 6 and 24 months
of age. These data indicate that the loss of
AdoA1R function in aging hearts is probably not
caused by decreased G-protein levels.
Our studies have shown that decreased AdoA1R
function in the senescent rat heart is not caused by changes in
AdoA1R number or to changes in
Gi levels, but rather by alterations in
receptor/G-protein coupling. The state of receptor/G-protein coupling
determines the affinity of the receptor for agonists such as SPA. Thus,
the percentage of high-affinity receptor sites is a reflection of the
number of receptors precoupled to G-proteins. The age-related decrease
in high-affinity receptors suggests a decrease in the number of
receptors precoupled to G-proteins. Consistent with this observation
Cai et al. (1997) have shown age-related decrease in the
ability of SPA to induce coupling of Gi3
and Go to the
AdoA1R. Thus, age attenuates precoupling and
receptor-stimulated coupling of AdoA1R. Thus, DR
attenuates age-related changes that potentially could lead to the
decrease in AdoA1R coupling.
Other studies also suggest that DR can attenuate age-related changes in
receptor function. Dietary restriction prevents the age-related
increase in rat liver beta receptors (Katz 1988; Dax et al., 1989). Dietary restriction alters or attenuates the
age-related decline alpha-1 adrenoceptor responsiveness in
various tissues (Gurdal et al., 1995; Chen et
al., 1997). Chen et al. (1997) showed that the effect
of DR is the result of altered G-protein binding to rat parotid cell
membrane. Although it is clear from our data and others that DR affects
receptor/G-protein coupling, the mechanism for the decrease is not
clear. DR may exert its effect by altering membrane fluidity (Benedetti
et al., 1988) or by preventing peroxidative damage to
membranes (Viani et al., 1991).
Our results also indicate that DR reverses the decrease in
beta adrenergic receptor function. It is well established
that there is an age-related decrease in beta adrenergic
receptor-mediated stimulation of adenylyl cyclase activity (O'Connor
et al., 1981, 1983; Narayanan and Derby, 1982) and of
cardiac contraction (Abrass et al., 1982). However, there
are only a few reports on the effect of DR on beta
adrenergic responses (Herlihy and Kim, 1994). In young adult rats, DR
increased the inotropic response of electrically driven left atria to
ISO; however, no senescent animals were examined in this study
(Herlihy, 1984). Scarpace and Yu (1987) showed that DR enhanced the
ISO-stimulated adenylyl cyclase activity from lungs of both young and
old rats. Similar to what has been suggested regarding the
AdoA1R by this manuscript, it has been proposed that the age-related decrease in beta adrenergic receptor
function is caused by a failure to form high-affinity agonist-receptor complexes (Narayanan and Derby, 1982; Scarpace and Abrass, 1986), a
decrease in the amount of adenylyl cyclase (Scarpace, 1990; Shu and
Scarpace, 1994) and/or decreased receptor coupling to G-proteins
(Insel, 1993; Gurdal et al., 1995).
Dobson et al. (1990) and Romano and Dobson (1996) have
proposed that an increase in the antiadrenergic actions of adenosine is
responsible for the age-related decrease in beta adrenergic receptor-mediated responses in the heart. In our experiments, endogenous adenosine is removed by adding adenosine deaminase; therefore, the decrease in beta adrenergic-mediated
stimulation of adenylyl cyclase in senescent rat hearts is most likely
not caused by excessive adenosine. In fact our data suggest some type of equilibrium that may mitigate age-related decreases in
beta adrenergic-stimulated adenylyl cyclase. Thus, the
ultimate increase in adenylyl cyclase within heart tissue may be the
same because there is also less inhibition by
AdoA1R. On the other hand, there are redundant
mechanisms for the inhibition of cyclase such as stimulation of
muscarinic receptors that do not decrease with age (Gao et
al., 1997). In this case there would be less increase in cyclase
in the senescent rat.
In summary, the present study provides evidence that ISO- and forskolin-stimulated cAMP production and AdoA1R-mediated antiadrenergic responses decline with age in the male rat heart and that these changes occur as early as 12 months of age. The age-related decline in AdoA1R-mediated antiadrenergic responses are not caused by changes in receptor density, but may be caused by decreases in receptor G-protein coupling. Dietary restriction attenuated the age-related decline in beta adrenoceptor-mediated and forskolin-stimulated activation of adenylyl cyclase and AdoA1R-mediated antiadrenergic responses. Our results indicate that DR maintains AdoA1R function during aging by maintaining receptor/G-protein coupling.
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Acknowledgments |
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N6-p-Sulfophenyladenosine was provided by Research Biochemicals International (Natick, MA) as part of the Chemical Synthesis Program of the National Institute of Mental Health, Contract N01 MH30003.
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Footnotes |
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Accepted for publication December 10, 1997.
Received for publication June 18, 1997.
1 This study was supported in part by grants from the National Institutes of Health (KO7 AG 00532, AG 11060) and the Allegheny Health Education Research Foundation.
Send reprint requests to: Dr. Joel Horwitz, Department of
Pharmacology, Allegheny University of the Health Sciences, MCP Hahnemann School of Medicine, 3200 Henry Ave., Philadelphia, PA
19129.
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Abbreviations |
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DR , diet restriction;
AL, ad
libitum;
AdoA1R, adenosine A1 receptor;
ISO, isoproterenol;
SPA, N6-p-sulfophenyladenosine;
8-SPT, 8-p-sulfophenyltheophylline;
3H-DPCPX, cyclopentyl-1,3-dipropylxanthine, 8-[dipropyl-2,3-3H],
Gpp(NH)p-5'-guanylylimidodiphosphate;
EGTA, ethyleneglycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid;
EDTA, ethylenediaminetetraacetic acid;
HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;
ANOVA, analysis of
variance;
CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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-adrenergic receptors in young and old rats.
J Gerontol
37:
156-160[Medline]. protein coupling in rat cardiac ventricles during aging.
Circ Res
81:
1065-1071.-adrenergic contractile response in aged hearts.
Circ Res
66:
1381-13901-adrenoceptor mediated responses during aging in Fischer 344 rats.
J Gerontol Biol Sci
50A:
B67-B71[Abstract]. subunits in the aging cardiovascular system.
J Gerontol
50A:
B14-B19.-adrenergic-sensitive adenylate cyclase in rat liver during aging.
Am J Physiol
254:
E54-E62-adrenergic receptors of myocardial membranes in aging: impairments in agonist-receptor interactions and guanine nucleotide regulation accompany diminished catecholamine-responsiveness of adenylate cyclase.
Mech Ageing Dev
19:
127-139[Medline].This article has been cited by other articles:
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P. Abete, G. Testa, N. Ferrara, D. De Santis, P. Capaccio, L. Viati, C. Calabrese, F. Cacciatore, G. Longobardi, M. Condorelli, et al. Cardioprotective effect of ischemic preconditioning is preserved in food-restricted senescent rats Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H1978 - H1987. [Abstract] [Full Text] [PDF]
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9-10
) and absence (
) of 100 µM Gpp(NH)p. Data are
mean ± S.E.M of seven animals from each group.