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Vol. 293, Issue 2, 599-606, May 2000
Department of Biology, Northwest Nazarene University, Nampa, Idaho (C.L.K.); Clinical Pharmacology and Gerontology Research Unit, Department of Veterans Affairs Medical Center, Boise, Idaho (S.C.M., R.V.M., S.M.J., R.D.O., R.E.V.); Mountain States Medical Research Institute, Boise, Idaho (C.L.K., S.C.M., R.V.M., R.D.O., R.E.V.); and Departments of Medicine and Pharmacology, University of Washington School of Medicine, Seattle, Washington (S.M.J., R.D.O., R.E.V.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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To characterize age-related changes in 
-adrenergic responsiveness
and to test the hypothesis that an increase in the effects of adenosine
contribute to impaired -adrenergic responsiveness, Fischer 344 rat
right atria (RA), left atria (LA), and left ventricular trabeculae
carnae were exposed to the -receptor agonist isoproterenol (ISO),
followed by four doses of the selective adenosine A1
receptor agonist cyclopentyladenosine (CPA). Spontaneous contractile
rates of adult RA were inhibited more than senescent RA by CPA.
Contractility (+dF/dt) of adult LA was reduced more than senescent LA
by CPA. Left trabeculae carnae tissue responded weakly to CPA, but
senescent tissue was less responsive than adult tissue. Senescent
atrial A1 receptor density was 56% greater than in adult
tissue, whereas the density in senescent ventricles was 39%
lower than in adult tissue. No significant difference in antagonist
affinities (Kd) of A1 receptor
was observed between adult and senescent atria. In addition, agonist
competition curves indicated a significant increase in senescent atrial
and a decrease in senescent ventricular tissue in the affinity of
agonist for high-affinity A1 receptors with no difference
in dissociation constant (Ki). No
significant age-related differences in atrial or ventricular tissues
occurred in either the antagonist affinity
(Kd) or density
(Bmax) of the -adrenergic receptors. CPA
was found to inhibit ISO-stimulated adenylate cyclase activity more in
senescent than in adult atrial and ventricular membrane preparations.
We conclude that age-related differences in functional response to ISO
and CPA, A1 receptor density, and ISO-stimulated adenylate
cyclase activity differ in atrial and ventricular myocardium.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Aging
is associated with known changes that occur in the cardiovascular
system of mammalian species. A decrease in -adrenergic-mediated inotropic, chronotropic, and vasodilating cardiovascular responses occurs with aging in both experimental animals and humans. Experimental animals have shown age-related changes in calcium handling and contractile function (Lakatta and Yin, 1982
). Heart rate
response to infused isoproterenol (ISO) is diminished in elderly human subjects (Vestal et al., 1979). Developed tension in isolated cardiac
tissue from rats exposed to ISO is reduced with age (Guarnieri et al.,
1980), and the vasodilatory effect of ISO on peripheral veins is
attenuated in older human subjects (Pan et al., 1986).
Adenosine, an endogenous nucleoside that is formed by the metabolism of
its corresponding nucleotides (ATP and ADP), has modulatory effects on
-adrenergic function. The adenosine response is mediated by two
extracellular receptors designated A1 and
A2, which are linked to adenylate cyclase (AC)
activity and the production of cAMP (van Calker et al., 1979; Londos et
al., 1980). Adenosine acts in a cardioprotective manner by directly
activating potassium channels in atria and the atrioventricular and
sinus nodes (Yatani et al., 1988; Kirsch et al., 1990).
Adenosine also can inhibit -adrenergic-stimulated AC (Romano and
Dobson, 1996).
During stress or exercise, cardiac performance is enhanced due to
release of catecholamines that interact with -adrenoceptor to
activate AC. This results in an increase in cAMP and phosphorylation of
cardiac proteins catalyzed by cAMP-dependent protein kinase. By way of
receptor-coupled inhibitory G-protein activation, adenosine has
inhibitory effects on -adrenergic receptor stimulation. Adenosine analogs impair both chronotropic and inotropic responses to
-adrenergic stimulation in isolated cardiac preparations from
animals (Dobson et al., 1986; Montamat et al., 1996). Dobson et al.
(1990, 1993) have shown that coronary adenosine release and cardiac
tissue interstitial adenosine concentration are enhanced in aged
myocardium compared with young adult heart, and that adenosine
antagonists partially correct the impairment of myocardial contractile
response to catecholamines. These studies suggest that adenosine
modulation of -adrenergic function may account for decreased
contractile responses to catecholamines in aged cardiac tissues.
To further characterize age-related changes in -adrenergic
responsiveness related to adenosine modulation and to test the hypothesis that an increase in the effects of adenosine contribute to
impaired -adrenergic responsiveness, isolated cardiac tissues from
young adult and senescent Fischer 344 (F344) rats were stimulated with
ISO followed by administration of cyclopentyladenosine (CPA), a
selective A1 receptor agonist. To characterize
age-related changes in the adenosine A1 and
-adrenergic receptors, radioligand receptor-binding studies were
conducted. In addition, to assess the influence of aging on
adenosine-mediated receptor-effector coupling, AC activity was measured
in crude atrial and ventricular membranes obtained from adult and
senescent F344 rats, an established model for aging research (Hazzard
et al. 1992).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals.
F344 rats aged 6 to 8 months (adult) and 23 to 24 months (senescent) were obtained from Harlan Industries (Indianapolis,
IN) under contract with the National Institute on Aging. The senescent animals were older than the age at which 50% mortality occurs (F344
rats median life span is 22 to 23 months according to the National
Research Council, 1981). On arrival, rats were examined and housed in
groups of two to four in 18- × 10-in. clear polycarbonate cages.
All animals were maintained on Wayne's Lab Blox F-4 ad libitum with
12-h light/dark cycle. Rats were used within 4 weeks of their arrival
and after 1 week of observation. Animals that exhibited signs and
symptoms of illness were not used in the study. The animals were cared
for in accordance to the guidelines outlined in the Guide for the Care
and Use of Laboratory Animals, and the protocol was approved by the
Institutional Animal Care and Use Committee. On the day of study,
animals were euthanized by decapitation, hearts were quickly removed,
and the left atrium (LA), right atrium (RA), and left trabeculae carnae
(LTC) were dissected from each heart.
Functional Studies. The isolated cardiac tissues were attached to an isometric force transducer (Kulite BG25) and suspended in a 15-ml muscle bath containing Krebs-bicarbonate buffer (pH 7.4) maintained at 30°C. The buffer consisted of the following components: 127 mM NaCl, 2.3 mM KCl, 2.5 mM CaCl2 (1 mM for LTC), 24 mM NaHCO3, 1.3 mM KH2PO4, 0.6 mM MgSO4, and 5.5 mM glucose. The tissue in the bath was continuously bubbled with 95% O2, 5% CO2. The muscles were stabilized over a period of 2 h before any measurements were recorded.
The RA were allowed to beat spontaneously at their own rate. The LA and LTC were stimulated electrically via platinum electrodes by 3-ms square pulses at a voltage 20% higher than the threshold strength delivered to the tissue at the rate of 30 contractions/min. Before beginning the protocol, LTCs were gradually stretched until a maximal contractile force was obtained (LMAX). They then were allowed to stabilize for another 45 min. Adenosine deaminase (ADA; 3 U/ml; Boehringer-Mannheim, Indianapolis, IN) was added during the last 15 min of the stabilization period. This concentration of ADA had no effect on the measured parameters (Table 1). ADA was purified by dialysis in phosphate buffer at pH 7.0 for 12 to 16 h before use (Dobson, 1983), and ADA activity was quantified by
spectrophotometric determination (Bergmeyer, 1983). Adult heart tissues
were exposed to the -adrenoceptor agonist, ISO
(10
8 M). To obtain a similar increase in
contractility, senescent cardiac tissues were exposed to a higher dose
of ISO (107 M). At maximal contraction, a
cumulative concentration response curve to CPA was obtained. CPA was
dissolved in 1.0 mM HCl to enhance solubility, and appropriate vehicle
control experiments were preformed.
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Antagonist Binding.
Radioligand-binding experiments were
conducted as previously described (Musser et al., 1993). Atrial
and ventricular tissue from adult and senescent male F344 rats were
obtained fresh and placed in 25 mM imidazole buffer, pH 7.4, containing
0.32 M sucrose, 1 mM NaEDTA, 10 µM phenylmethlsulfonyl fluoride
(PMSF), 0.7 µg/ml pepstatin, and 0.5 µg/ml leupeptin (pepstatin and
leupetin from Boehringer-Mannheim). The tissue was homogenized for
15 s with a Polytron homogenizer with a setting of 6. The
homogenate was centrifuged at 1000g for 10 min. The
supernatant was filtered through gauze and diluted with an equal volume
with 25 mM imidazole buffer containing 1 mM NaEDTA, 10 µM PMSF, 0.7 µg/ml pepstatin, and 0.5 µg/ml leupeptin. The supernatant was
centrifuged at 48,000g for 30 min and the pellet was used
immediately for the binding studies.
-adrenergic receptor studies, the pellet was resuspended in 25 mM imidazole buffer, pH 7.4, containing 5 mM
MgCl2 to a final protein concentration of 100 µg/ml. The reconstituted pellet was incubated with 0.12 mg/ml BSA
before the reaction. The nonselective antagonist -adrenoceptor
antagonist (
)-3-[125I]iodocyanopindolol
([125I]CYP; Amersham) was used at concentration
of 3 pM to 3 nM to construct saturation isotherms. The reactants were
incubated for 90 min at 37°C before vacuum filtration. Nonspecific
binding was defined as that occurring in the presence of 1 µM
propranolol. The filters were cut and counted with a gamma counter.
Protein concentrations were determined by the method of Bradford
(1976). An iterative nonlinear curve fitting program ReceptorFit
Saturation Two-Site (Lundon Software, Inc., Chagrin Falls, OH) was used
to determine the Kd and
Bmax for each isotherm.
AC Inhibition. Adult and senescent F344 rats were sacrificed, the hearts rapidly removed, perfused retrograde with 20 ml of ice-cold normal saline to remove blood, and the atria carefully separated from the ventricular tissue. A single heart was used for each assay of AC activity in ventricle and three (senescent) or four (adult) hearts were pooled for determination of atrial AC activity.
Tissue was gently homogenized in ice-cold 10 mM imidazole buffer containing 0.27 M sucrose, 1 mM dithiothreitol, 0.1 mM benzamidine, 10 mM disodium EDTA, 10 µM PMSF, and 0.5 µg/ml leupeptin. The homogenate was centrifuged at 750g for 10 min, the supernatant filtered through four layers of moist gauze, centrifuged at 45,000g for 20 min, and the pellet resuspended in 4 ml of the same buffer but without sucrose. After removal of samples for protein determination, the resuspended pellet was incubated at room temperature for 30 min with ADA (7.5 U/ml) and 0.001% SDS. AC activity was determined in duplicate 50-µl aliquots of homogenate in a total volume of 150 µl of 50 mM imidazole buffer containing 100 mM NaCl, 200 µM papaverine, 100 µM GTP, 150 µM dATP, 150 µM cAMP, 0.1 mg/ml bacitracin, 100 µM dithiothreitol, 1 µM CaCl2, 5.5 mM KCl, 3 mM MgCl2, 43 U/ml creatine phosphokinase, 0.75 mg/ml creatine phosphate, and 0.5 × 106 cpm [32P]ATP. To eliminate the effect of adenosine produced during the reaction, cardiac membranes were preincubated with ADA (7.5 U/ml) and [
-32P]ATP was used as the
substrate. The production of [32P]cAMP with
recoveries based on [3H]cAMP content was
determined as described by Halvorson and Nathanson (1984). Samples were
purified and separation of cAMP was accomplished by using the modified
method of Salomon (1979). All chemicals and reagents not specified were
obtained from Sigma Chemical Co. (St. Louis, MO) and were of best
analytical grade available.
Data Analysis. The results of the isolated atrial and ventricular functional and AC studies were analyzed statistically by either one- or two-way ANOVA as appropriate. The Student-Newman-Keuls method for multiple mean comparison was used for post hoc analysis. Results are expressed as mean ± S.E. unless otherwise noted. Statistical comparisons of the resulting receptor binding studies between young and senescent groups were made with the unpaired Student's t test.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolated Cardiac Function Studies.
The baseline spontaneous
contraction rate in senescent RA was significantly lower than in adult
RA (P < .01). Addition of ISO significantly increased
the spontaneous contraction rate in RA from both adult and senescent
rats. Although the maximal response to ISO was less in senescent than
in adult RA (Table 1), the difference was not significant. Furthermore,
the change from basal contraction rate was similar in both groups (Fig.
1). Cumulative administration of CPA to
spontaneously beating RA pretreated with ISO resulted in a
concentration-dependent decrease in contractile rate (Fig. 1). The
effects of CPA on RA were calculated as differences between the drug
and baseline responses. Although CPA inhibited the increase in
contraction rate induced by ISO in both age groups, there was a
significant interaction between age and concentration of CPA
(P < .05). This indicates that the overall rate of
change in the contraction rate in response to inhibition by CPA was
greater in adult than in senescent preparations.
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A1 Receptor Binding Characteristics. [3H]DPCPX saturation-binding isotherms were constructed to characterize A1 receptor binding in crude adult and senescent F344 atrial and ventricular membrane preparations. Antagonist binding was saturable and concentration dependent. Scatchard plots of the data were linear, suggesting a single homogenous class of binding sites. In atria there was a significant age-dependent increase (55.5%) in receptor density (B max) from 22.5 to 35.1 fmol/mg protein (P < .05) with no significant change in the receptor affinity (Kd) (Table 2). In contrast, the ventricular data displayed an opposite change with a 38.5% decrease in receptor density (B max) from 9.2 fmol/mg protein in the young adult to 5.6 fmol/mg protein in the senescent rat (P < .005) with no significant change in the receptor affinity (Kd) (Table 2).
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-Adrenergic Receptor-Binding Characteristics.
To
investigate the -adrenergic receptor characteristics of the young
adult and senescent rat atria and ventricles, saturation-binding isotherms were constructed with the antagonist
[125I]CYP. Neither atrial nor ventricular data
showed any changes in the Bmax or
Kd with age (Table 2).
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Inhibition of AC Activity.
Basal AC activity was significantly
reduced (P < .05) in senescent atria (49%) and
ventricles (32%) compared with adult myocardial tissue (Table
4). A significant reduction with age in
stimulation of AC activity by ISO, forskolin, and
guanosine-5'-(,
-imido)- triphosphate [Gpp(NH)p] was observed in
both atria and ventricles (Table 4). The ability of Gpp(NH)p to
stimulate AC also was significantly reduced in senescent atria and
ventricles. Forskolin (a direct activator of AC catalytic subunit) and
Gpp(NH)p exhibited significantly greater AC activity in ventricles than
in atria (Table 4).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Recently, several studies have examined the reduced responsiveness
of aged myocardium in relation to adenosine (Dobson et al., 1990;
Mudumbi et al., 1995; Montamat et al., 1996; Romano et al., 1996; Gao
et al., 1997). The involvement of adenosine in this process
is supported by the observation that the reduced responsiveness of
adenosine receptor stimulation in aged heart is not observed when
theophylline, an adenosine antagonist, is present (Londos et al., 1978;
Dobson, 1983).
Earlier studies have investigated the effects of adenosine after ISO
stimulation on isolated perfused aged hearts (Dobson et al., 1990).
Although these studies show age-related alterations due to
adenosine, they do not examine the effects of adenosine on isolated
cardiac muscle tissues. In contrast, our functional studies examine the
effects of CPA, a selective A1 adenosine receptor agonist, on individual heart tissues. The results demonstrate age
differences in the functional response to CPA, in the number of
A1 receptors, and in AC activity when
-adrenoceptors have been activated by ISO. This is particularly
noteworthy in the absence of apparent age-dependent down-regulation of
-adrenergic receptors as indicated by the absence of significant
changes in ISO-stimulated contractile rate response and +dF/dt, as well
as Bmax.
The contribution of the LA contraction to diastolic ventricular filling
becomes greater as humans age (Miyatake et al., 1984; Sartori et al.,
1987). Modulators such as adenosine may be important in regulating
cardiac function with age. LA contractility (+dF/dt) and RA contraction
rate of tissue from senescent F344 rats are less responsive to
inhibition by CPA than that obtained from young animals. In
contrast, our binding studies indicate an increase in the density of
A1 receptors in atria with age, although there is
no significant change in Kd.
Competition-binding experiments indicate that as the atria age, there
is a shift from single-site binding to two-site binding with 74% of
the sites being characterized as high-affinity binding sites with a
lower Ki (8.93 to 1.71 nM). Due to an
increased 5' nucleotidase activity in the senescent heart (De Tata et
al., 1985), an increased adenosine production in the senescent heart
(Dobson et al., 1990; Dobson and Fenton, 1993), and increased atrial
A1 receptors with age, an increase in the
response to adenosine within the cell might be expected. Yet, examining
receptor function, we found a decrease in basal AC activity as well
as forskolin- and Gpp(NH)p-stimulated AC activity, indicating a
decline in receptor-effector coupling with age.
Our functional experimental results (contraction rate, contractility,
and 90% RT) seem to indicate that even with increased adenosine in the
atrial tissue as reported by others (Dobson et al. 1990; Dobson and
Fenton, 1993), as well as increased receptor density and increased
specific binding, there appears to be a defect either in signal
transduction in the cell membrane or in one or more subsequent steps in
the effector pathway. The latter could involve the interaction of the
receptor with its GTP-binding protein (G protein) or the interaction of
the G-protein subunit and AC itself, resulting in decreased AC activity
and functional response. The 90% RT of adult LA was a higher
percentage of their ISO response than that of senescent LA. This effect
may be due to changes in the calcium sequestration with aged
muscle cells (Lakkata, 1987).
Miyamoto et al. (1994) and more recently by Cai et al.
(1997) have investigated G-protein coupling in the heart as it
relates to aging. Studying -adrenoceptor/G-protein coupling,
Miyamoto et al. (1994) found in aged Wistar rat ventricle that there
was a decrease in three Gs
subunits with no
change in Gi, Gq,
Go, or Gcommon
. They
also found a corresponding decrease in Gs
mRNA. This suggests a change in the cardiac excitation and contraction
coupling after -adrenoceptor stimulation, which are mediated in part
by Gs. Cai et al. (1997) also investigated the
G proteins associated with adenosine receptors. A1 receptors have been shown to couple to
Gi1, Gi2,
Gi3, and Go in
reconstituted systems (Munshi et al., 1991; Jocker et al., 1994; Figler
et al., 1996). Using F344 rats, Cai et al. (1997) found a decrease in
precoupling to Gi3 and Go
(activation in the absence of agonist activation) as well as a decrease
in agonist-stimulated protein coupling of A1
receptors to Gi3 and Go.
This loss of A1 receptor function appears to be
associated with a receptor/G-protein uncoupling in aged ventricles.
Functionally, adenosine has much smaller effects in ventricular tissue than in atrial tissue. In this study, the effect of CPA on LTC was small, but at the same dose the response of adult tissue was significantly greater than that of senescent tissue. Thus, the response to receptor stimulation with the A1 receptor agonist CPA decreased with age. This may be due at least in part to age differences in receptor characteristics. Both the number of A1 receptors and the antagonist Kd decreased significantly with age. Agonist competition experiments in ventricles indicate a predominance (76.6%) of high-affinity receptors, whereas there was a nearly equal distribution of high- and low-affinity sites (56.1 and 43.9%, respectively) in the senescent animals.
Gao et al. (1997) found that there was no difference in basal AC
activity in crude cardiac ventricular membranes from 1-, 6-, and
24-month F344 rats. Our results, however, show that basal AC activity
in ventricular tissue decreased significantly with age (Table 4). One
possible reason for this disparity is that we used fresh tissue for our
assay, whereas Gao et al. (1997) used tissue that had been frozen.
ISO-stimulated and forskolin-stimulated ventricular AC activity
decreased with age, confirming the findings by others (Dobson et al.,
1990; Gao et al., 1997). The experiments with Gpp(NH)p-stimulated AC
activity also indicate a decreased AC activity in ventricular tissue
with age. Our data confirm previous studies with -adrenergic
antagonists that showed no significant changes in -adrenergic
receptor characteristics with increasing age (Newman et al., 1989,
Dobson et al., 1990; Scarpace, 1990; Shu and Scarpace, 1994). Shu and
Scarpace (1994) also found that the -subunits of
Gs and Gi remain unchanged
with age in F344 rats, but there is a decrease in forskolin-stimulated
AC activity with a corresponding decrease in forskolin-binding sites
with age (sensitivity was unchanged). Our results indicate that the decreased functional response in ventricular tissue may be due to a
decreased receptor population. The basal activity of AC was higher in
ventricles than atria. In addition, we found that -adrenergic stimulated AC activity also was reduced in atrial tissue (Table 4).
Cardiac sensitivity to -adrenergic stimulation decreased significantly with age in atrial and ventricular tissues.
Although there were changes in A1 receptor
density, AC activity was less in senescent than adult tissue. These
changes may be due to changes in receptor/G-protein coupling.
In summary, the age-related differences in functional responses to ISO and CPA, A1 receptor density, and ISO-stimulated AC activity differ in atrial and ventricular myocardium. If similar tissue-specific differences exist in the hearts of other species including humans, they will influence and thus complicate the interpretation of the responses to endogenous adenosine and to exogenous adenosine receptor agonists and antagonists.
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Acknowledgments |
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We thank Beth Musser, Donna McDonald, Jason Sandidge, and Amber Overton for excellent technical assistance.
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Footnotes |
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Accepted for publication January 23, 2000.
Received for publication September 8, 1999.
1 This study was supported by the Department of Veterans Affairs (Office of Research and Development, Medical Research Service), National Institutes of Health Grants AG00525 and AG09559, Northwest Nazarene College, and the Mountain States Medical Research Institute.
Send reprint requests to: Robert E. Vestal, M.D., Research Service (151), VA Medical Center, 500 W. Fort St., Boise, ID 83702. E-mail: rvestal{at}micron.net
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Abbreviations |
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ISO, isoproterenol;
AC, adenylate cyclase;
CPA, cyclopentyladenosine;
LA, left atria;
RA, right atria;
LTC, left
ventricular trabeculae carnae;
ADA, adenosine deaminase;
RT, relaxation
time;
TTPF, time to peak force;
PMSF, phenylmethylsulfonyl fluoride;
[125I]CYP, ()-3-[125I]iodocyanopindolol;
[3H]DPCPX, 1,3-[3H]dipropyl-8-cyclopentylxanthine;
Gpp(NH)p, guanosine-5'-(,-imido)triphosphate;
CB, carbochol.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-adrenergic induced responses in aged hearts.
Am J Physiol
265:
H494-H503-adrenergic stimulation of the aged heart.
Am J Physiol
239:
H501-H508.-adrenoceptors in mammalian atrial and ventricular myocardium.
Eur J Pharmacol
246:
105-111[Medline].-adrenoceptor sensitivity in the elderly.
Clin Pharmacol Ther
26:
181-186[Medline].-subunits.
Nature (Lond)
336:
680-682[Medline].
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