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INTRODUCTION |
Cardiac contractility is controlled by several G protein-coupled
receptors (GPCRs),1 such as
1- and 2-adrenergic receptors
(1ARs and 2ARs), that function through
stimulatory GTP-binding regulatory proteins (G
s) and
activate adenylyl cyclase (AC). Activation of AC increases the
formation of cAMP, which activates cAMP-dependent protein kinase A resulting in the phosphorylation of proteins controlling cardiac excitation-contraction (1). An important recent discovery is
that 2ARs (but not 1ARs) in both rat (2,
3) and human heart (4) also activate Gi, a G-subunit
that inhibits AC (5). We also demonstrated that Gi
couples to several other Gs-coupled receptors in human
heart, including receptors for histamine, glucagon, and serotonin (4).
Coupling of 2AR and other Gs-coupled
receptors to Gi is relevant to cardiac function because
inactivation of Gi by pertussis toxin (PTX) increases myocyte contractility in rat heart (6) and increases both basal and
receptor-mediated AC activity in human heart (4). In addition to
inhibiting AC, the Gi pathway in heart is involved in
anti-apoptotic effects (7-9).
The dual coupling of 2AR to both Gs and
Gi is likely to alter AR function in diseases in
which cardiac Gi levels are increased, such as
congestive heart failure and hypertensive cardiac hypertrophy
(10-13). Indeed, both congestive heart failure and cardiac hypertrophy
are characterized by a reduced responsiveness of ARs. Similarly, a
decline in the responsiveness of cardiac ARs due to aging has been
demonstrated in both humans (14-17) and rodents (18-21). The
age-induced decrease in AR responsiveness is characterized at the
molecular level by decreased stimulation of AC and at the whole organ
level by a decrease in heart rate, ejection fraction, and cardiac output.
There have been conflicting reports about the effect of age on cardiac
Gi levels in both humans and rodents. In one study of
human heart, Gi levels were measured in atrial
appendages received from surgical patients, and it was found that
Gi expression increased with age (17). Another study
examined Gi expression in human ventricles from hearts
that were not suitable for transplant and found no change in
Gi with age (16). Similar studies in rat heart have
yielded inconsistent results even when the same strain of rat was used.
In Fisher 344 rats, Roth et al. (21) reported an
age-associated increase in cardiac Gi, which is reduced by chronic dynamic exercise. Johnson et al. (22) found an
increase in Gi mRNA but no change in
Gi protein. Two other reports found no increase in
cardiac Gi protein (23, 24). In Sprague-Dawley rats,
Bohm et al. (19) found that age increases cardiac
Gi, and the increase in Gi is reduced by
exercise. In Wistar rats, Bazan et al. (25) reported an
increase in cardiac Gi with age, but Xiao et
al. (6) and Miyamoto et al. (26) found no change. Some
of the reported differences on the effect of age may be attributable to
experimental design. For example, the finding of Chin et al. (24) that age does not increase cardiac Gi in Fisher 344 rats may be explained by the fact that these investigators used
16-month-old rats for their old age group versus
24-month-old rats used by others. Moreover, most studies examined
Gi expression by immunoblotting only, and different
Gi antibodies were used. Nevertheless, the available
evidence favors an increase in Gi with age, as indicated by the recent review of Roka et al. (27). However, two
recent reviews by Lakatta (28, 29) on global changes in cardiovascular aging state that Gi in heart does not increase with age.
We believe that this conclusion is premature.
The importance of Gi in cardiac function underscores the
need to establish whether or not cardiac Gi is affected
with age. Therefore, we undertook a detailed study on the effect of age on Gi expression in rat ventricular membranes. We used
Fisher 344 rats because these rats have been the most widely used rat strain for aging studies (30), and age-induced changes in cardiac structure have been characterized (31). Expression of the predominant cardiac subtypes of Gi, as well as of Gs,
Go, and the major subtypes of G, was assessed by
immunoblotting. Levels of PTX-sensitive Gi/Go proteins were also examined by
radiolabeling through PTX-catalyzed ADP-ribosylation. In addition,
Gi2 activity was assessed using photoaffinity labeling
with [32P]azidoanilido-GTP (AA-[32P]GTP).
We show age-dependent increases in both Gi2
expression levels and activation of Gi2 by
2AR and other G protein-coupled receptors in heart. The
age-induced increase in Gi2 has the functional effect of
suppressing AC activity, which is restored by disrupting receptor-Gi coupling with PTX.
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EXPERIMENTAL PROCEDURES |
Materials--
R(
)-isoproterenol-(+)-bitartrate,
ICI 118,551, and CGP 20712A were obtained from Research Biochemicals
International (Natick, MA). Glucagon was from Peninsula Laboratories
(San Carlos, CA). Forskolin, carbachol, alprenolol, and Ponceau S were
from Sigma. [-32P]ATP, [-32P]GTP,
[3H]cAMP, ()-[125I]iodocyanopindolol
([125I]CYP), RM/1 antibody specific for Gs
raised against the peptide sequence RMHLRQYELL, and AS/7 antibody
specific for Gi1 and Gi2 raised against
the peptide sequence KNNLKDCGLF, were from PerkinElmer Life Sciences.
Antibody specific for Gi1 and Gi3,
antibodies specific for G1 and G2, and
goat anti-rabbit IgG secondary antibody conjugated to horseradish
peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA). GK-1
antibody for Go was prepared by our laboratory and was
characterized previously (32). Recombinant G protein standards from
Escherichia coli were from Calbiochem (La Jolla, CA).
Protein A-Sepharose and [32P]nicotinamide adenine
dinucleotide ([32P]NAD) were from Amersham Biosciences.
PTX was from List Biological Laboratories (Campbell, CA).
SuperSignal® chemiluminescent reagent was from Pierce.
Animals--
Sixteen Fisher 344 rats (eight 3-month-olds and
eight 24-month-olds) were obtained from the National Institute on Aging
under an Institutional Animal Care and Use Committee-approved protocol. Animals were sacrificed by decapitation, and left ventricles were extracted immediately, frozen in liquid nitrogen, and stored at 80 °C.
Membrane Preparation--
Samples were thawed, then homogenized
for 25 s in a Polytron PT3000 (Brinkmann Instruments, Westbury,
NY) at medium speed, in 20 mM Tris (pH 7.4), 2 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 5 µg/ml aprotinin. Membranes were pelleted by
centrifugation at 100,000 × g in an Optima TL
Ultracentrifuge (Beckman, Fullerton, CA). Membranes were resuspended,
repelleted, and then resuspended again in the appropriate final
resuspension buffer (see below) at ~2-4 mg of protein/ml. For
photoaffinity labeling assays, the final resuspension buffer contained
50 mM HEPES (pH 7.4), 1 mM EDTA, 50 mM NaCl, and 2 mM benzamidine. For
immunoblotting, ADP-ribosylations, and AC assays, the final
resuspension buffer contained 75 mM Tris (pH 7.4), 12.5 mM MgCl2, 2 mM EDTA, 1 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, 5 µg/ml aprotinin.
Immunoblot Analysis--
Immunoblotting was performed by
separating 10 µg of membrane protein by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by
electrophoretic transfer onto polyvinylidene difluoride membranes. The
membranes were stained for 5 min with 0.5% Ponceau S in 1% acetic
acid to check for equal protein loading and transfer. The membranes
were then blocked for 1 h with 10% nonfat milk in 20 mM Tris (pH 7.4), 500 mM NaCl, and 0.1% Tween 20, probed with either a 1:6000 dilution of RM/1, AS/7, or GK-1 antibody, a 1:200 dilution of anti-Gi3 antibody, or a
1:500 dilution of G antibodies, then incubated with a 1:2000
dilution of goat-anti-rabbit secondary antibody coupled to horseradish
peroxidase. The immunoreactive proteins were visualized by incubation
with SuperSignal® chemiluminescent reagent and exposure to x-ray
film. Protein bands were quantified by densitometry using Quantity One
software (Bio-Rad, Hercules, CA). Electrophoresis and subsequent
analysis of various amounts of protein yielded linear densitometric results.
Photoaffinity Labeling with
AA-[32P]GTP--
AA-[32P]GTP was
synthesized according to published procedures and purified by thin
layer chromatography on polyethylenimine cellulose (J.T. Baker,
Phillipsburg, NJ) (32, 33). Photoaffinity labeling of membranes (30 µg of protein) was performed using 2 µCi of
AA-[32P]GTP in a total volume of 60 µl in the presence
of various drugs indicated in the figure legends. Photolabeled G
subunits were separated and subsequently analyzed by
SDS-PAGE/autoradiography. The autoradiograms showed a prominent band
below the 45-kDa molecular mass marker that represented
Gi2 because it was immunoprecipitated with AS/7 antibody.
Pertussin Toxin Treatment of Ventricular Membranes for AC
Assays--
PTX (50 ng/µl) was activated by incubation with 100 mM DTT and 0.25% SDS for 30 min at 30 °C as described
(34). The activation reaction was stopped by the addition of four
volumes of 1 mg/ml ice-cold bovine serum albumin (BSA). Activated PTX
(1 ng/µl) was added to membranes (1.0-1.5 mg/ml protein) in a buffer
containing 37.5 mM Tris (pH 7.4), 6.25 mM
MgCl2, 1 mM EDTA, 5 mM NAD, 2.5 mM ATP, 4 mM GTP, 10 mM thymidine,
10 mM DTT, 0.005% SDS, 0.08 mg/ml BSA, 0.5 mM
benzamidine, 5 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin,
and 2.5 µg/ml aprotinin. The mixture was incubated for 30 min at
30 °C, then an equal volume was added of ice-cold 50 mM
Tris (pH 7.4) with 1 mM benzamidine, 10 µg/ml soybean
trypsin inhibitor, 10 µg/ml leupeptin, and 5 µg/ml aprotinin.
Membranes were pelleted by centrifugation at 100,000 × g in an Optima TL ultracentrifuge and resuspended in the
final resuspension buffer containing 75 mM Tris (pH 7.4),
12.5 mM MgCl2, 2 mM EDTA, 1 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 5 µg/ml aprotinin, at ~0.5-1.5 mg of
protein/ml. A second tube containing the same volumes of all
constituents, with the exception of H2O in place of PTX,
was treated in the same fashion and used as a control.
ADP-ribosylation of PTX-sensitive G Proteins--
PTX (50 ng/µl) was activated by incubation with 100 mM DTT and
0.25% SDS for 30 min at 30 °C as described (34). The activation reaction was stopped by the addition of four volumes of 1 mg/ml ice-cold BSA. Activated PTX (1 ng/µl) was added to membranes
(1.0-1.5 mg/ml protein) in a buffer containing 37.5 mM
Tris (pH 7.4), 6.25 mM MgCl2, 1 mM
EDTA, 2.5 mM ATP, 4 mM GTP, 10 mM
thymidine, 10 mM DTT, 0.005% SDS, 50 µM NAD,
10 Ci/mmol [32P]NAD, 0.08 mg/ml BSA, 0.5 mM
benzamidine, 5 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin,
and 2.5 µg/ml aprotinin. The mixture was incubated for 30 min at
30 °C, and then 100 µl of sample buffer (22.5 mM Tris
(pH 6.8), 7.2% SDS, 9% glycerol, 0.01% bromphenol blue, and 10%
2-mercaptoethanol) was added to each tube, and samples were separated
by SDS-PAGE and exposed to autoradiographic film. A second tube
containing the same volumes of all constituents, with the exception of
H2O in place of PTX, was treated in the same fashion and
used as a control. Specific phosphorylation bands were identified on
autoradiograms of the dried gels. 32P incorporation was
quantified by excision of the radioactive bands, addition of 7 ml of
Lefkofluor scintillant (Research Products International, Mount
Prospect, IL), and counting in a liquid scintillation counter (Wallac
1409, EG&G Wallac, Gaithersburg, MD).
Adenylyl Cyclase Assays--
AC activity was measured according
to the method of Salomon and coworkers (35, 36) as detailed previously
(4). Briefly, 20 µl of control or PTX-treated ventricular membranes
(20-30 µg protein) were added into a total volume of 50 µl
containing 30 mM Tris (pH 7.4), 5 mM
MgCl2, 0.8 mM EDTA, 0.12 mM ATP,
0.06 mM GTP, 2.8 mM phosphoenolpyruvate, 50 µg/ml myokinase, 0.1 mM cAMP, 10 µg/ml pyruvate kinase,
0.4 mM 3-isobutylmethylxanthine, 1 µCi of
[-32P]ATP, and the indicated drugs. After incubating
the samples for 15 min at 37 °C, the reaction was terminated with
900 µl of stop buffer (360 µM ATP, 285 µM
cAMP, and 50,000 cpm/ml [3H]cAMP), and cAMP was isolated
by sequential chromatography over Dowex and alumina columns. 14 ml of
Lefkofluor scintillant was added to each sample, and
[32P]cAMP and [3H]cAMP were counted in a
liquid scintillation counter.
Radioreceptor Binding Assays--
AR density was determined
by [125I]CYP binding using a saturating concentration of
200 pM. Nonspecific binding of [125I]CYP was
determined by the inclusion of 1 µM alprenolol. Ligand binding was performed in triplicate (15 µg of membrane protein per
tube) in a final volume of 500 µl consisting of 75 mM
Tris (pH 7.4), 12.5 mM MgCl2, 2 mM
EDTA, 1 mM benzamidine, 10 µg/ml soybean trypsin
inhibitor, 10 µg/ml leupeptin, and 5 µg/ml aprotinin. The reaction
was incubated and agitated for 2 h at room temperature. Bound
[125I]CYP was separated from free [125I]CYP
by rapid vacuum filtration onto glass fiber (GF/C) filters (Whatman
International, Maidstone, UK). Filters were washed three times with 4 ml of ice-cold 50 mM Tris (pH 7.4) using a Brandel cell
harvester (Brandel, Gaithersburg, MD) and counted in a gamma counter
(Packard, Downers Grove, IL).
Statistical Analysis--
Results are presented as mean ± S.E. Each set of data was analyzed by Shapiro-Wilk tests for normality,
then the statistical significance of comparisons between data from
young and old samples was determined by performing unpaired Student's
t tests and exact Wilcoxon Mann-Whitney tests on the mean
values of each data set. p < 0.05 was considered
significant for all comparisons.
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RESULTS |
Expression of Gi2 in Rat Left Ventricular Membranes
Increases with Age--
Fig. 1 shows
immunoblots for several G and G protein subunits in left
ventricular membranes from young (3 months) and old (24 months) rat
hearts. We examined the expression of Gi2,
Gi3, Go, Gs,
G1, and G2, all of which previously have
been detected in rat heart (26, 37). Quantitation of bands by
densitometry reveals that, on average, there is a significant increase
of 58 ± 7% in Gi2 in old rat ventricles. In
contrast, immunoblotting of each of the other G protein subtypes
indicates that expression is unchanged between young and old rat
ventricles.
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Fig. 1.
Age increases
Gi2 in rat left ventricle.
Young (Y) and old (O) rat ventricular membranes
were separated by SDS-PAGE, transferred to polyvinylidene difluoride
membranes, probed with antibodies selective for G and G subunits,
and visualized by chemiluminescence as described under "Experimental
Procedures." n = 6 for each age group. Each tissue
was assayed at least twice, and representative immunoblots are
shown.
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PTX-sensitive G Proteins in Left Ventricular Membranes Increase
with Age--
To confirm the increase in Gi2 observed
by immunoblotting by another independent method, we examined
ADP-ribosylation of PTX-sensitive G proteins
(Gi/Go) in ventricles from young and old
rats. This method has been used previously by Feldman et al. (10) to demonstrate a Gi increase in the failing human
heart and by Bohm et al. (19) to demonstrate a
Gi increase in the hearts of old Sprague-Dawley rats. As
Fig. 2 shows, the amount of
Gi/Go labeled with [32P]NAD
in PTX-treated membranes is significantly increased by 39 ± 8%
in ventricles from older rats.
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Fig. 2.
Age increases levels of PTX-sensitive G
proteins. Young (Y) and old (O) rat
ventricular membranes were ADP-ribosylated by incubation with activated
PTX and [32P]NAD then subjected to SDS-PAGE followed by
autoradiography. The radioactive bands were excised and counted as
described under "Experimental Procedures." n = 6 for each age group. Each tissue was assayed twice, and a representative
autoradiogram is shown.
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More Gi2 Is Activated in Left Ventricular Membranes
from Older Hearts--
We next determined whether the increased
expression of Gi2 in older left ventricular membranes is
accompanied by increased activation of Gi2. To this end,
we assessed the effect of age on the ability of 2AR and
other GPCRs to stimulate photoaffinity labeling of Gi2
with AA-[32P]GTP. Activated GPCRs catalyze the exchange
of GTP for GDP on -subunits of G proteins associated with the GPCR,
so the amount of AA-[32P]GTP incorporated into the
-subunit gives a direct measure of the extent of G protein
activation. Fig. 3A shows that
stimulations through 2ARs and glucagon receptors are
significantly increased with age, from 102 ± 25% and 101 ± 31% above basal levels in young ventricles, to 226 ± 33% and
244 ± 40% above young basal levels, respectively. These results
indicate that, as age increases, more Gi2 is activated
upon stimulation of 2ARs and glucagon receptors. Basal
labeling of Gi2 also significantly increases (by 83 ± 18%) in older membranes (Fig. 3A), consistent with the
increased expression of Gi2 in older ventricles shown in
Fig. 1. Photoaffinity labeling of stimulatory Gs is not
altered with age (data not shown).
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Fig. 3.
Increased activation of
Gi2 proteins in old hearts.
A, photoaffinity labeling of old rat ventricular membranes
reveals greater basal, 2AR-stimulated, and glucagon
receptor-stimulated incorporation of AA-[32P]GTP into
Gi2 than in young ventricles. Membranes were
photolabeled with AA-[32P]GTP in the presence of no drug
(Basal), 100 µM isoproterenol
(Iso), or 50 µM glucagon (Gluc), as
described under "Experimental Procedures." The photolabeled
membranes were subjected to SDS-PAGE followed by autoradiography.
n = 6 for each age group, and a representative
autoradiogram is shown. Histogram data are expressed as percentage of
basal incorporation in young atria. *, p < 0.05 compared with same condition in young samples. B, muscarinic
receptor-induced photoaffinity labeling also reveals greater
incorporation of AA-[32P]GTP into Gi2 in
older ventricles. Membranes were photolabeled with
AA-[32P]GTP in the presence of no drug
(Basal), 100 µM isoproterenol
(Iso), or 100 µM carbachol (Carb),
as described under "Experimental Procedures." The photolabeled
membranes were subjected to SDS-PAGE followed by autoradiography.
n = 4 for each age group. A representative
autoradiogram is shown.
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We next determined whether activation of Gi2 is also
increased in aged heart through muscarinic acetylcholine receptor, a GPCR that interacts with Gi but not Gs.
As shown in Fig. 3B, stimulation of muscarinic receptors in
older left ventricular membranes produces greater photoaffinity
labeling of Gi2 with AA-[32P]GTP than in
young membranes.
AC Activity Is Decreased in Older Rat Ventricles--
Figs. 1, 2,
and 3 show increased Gi2 protein expression and
activation in old rat heart. Because stimulation of
Gi-coupled receptors inhibits AC, we determined whether
there are age-dependent increases in inhibition of cardiac
AC production of cAMP. As shown in Table
I, basal AC activity is significantly
lower in old hearts, as are stimulations by 2ARs
(isoproterenol + 1AR antagonist CGP 20712A) and glucagon
receptors. Age-dependent decreases in stimulations by
1ARs (isoproterenol + 2AR antagonist ICI
118,551) and forskolin are not significant. To ascertain that the
decrease in AC activity in older membranes was not because of a
decrease in receptor number, we examined the expression level of ARs
in both young and old ventricular membranes. We determined AR
density using a saturating concentration of [125I]CYP,
and found no difference in the mean density of ARs between young and
old rat ventricles (Table I). These results are similar to other
reports on AR density in Fisher 344 rat hearts (21, 38-42), though
Mader et al. (43) did report a decrease in affinity. In
other rat strains there may be moderate decreases in receptor number
(6, 19, 25, 44).
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Table I
AC activity and AR expression in young and old rat left
ventricular membranes
Receptors tested were 1ARs (measured by inclusion of 100 µM isoproterenol + 100 µM ICI 118,551 (2AR antagonist)), 2ARs (100 µM
isoproterenol + 100 µM CGP 20712A (1AR
antagonist)), glucagon receptors (50 µM glucagon) and
muscarinic receptors (100 µM carbachol (Carb)). Direct
activation of AC was tested with forskolin (50 µM).
n = 6 for each age group, AC assays were performed in
duplicate, and binding assays were performed in triplicate.
*, p < 0.05 compared with AC stimulation by
same condition in young membranes.
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We also examined whether there are age-dependent changes in
the ability of muscarinic receptors to inhibit 1AR- and
2AR-stimulated AC in rat ventricle. As shown in Table I,
stimulation of muscarinic receptors strongly inhibits AR-stimulated
AC in both young and old rat hearts. Because muscarinic receptors are
able to inhibit AC stimulation almost completely in these membrane
preparations, there is no significant difference between the maximal
percent inhibition in young and old hearts, despite the increased
coupling of muscarinic receptors to Gi2 shown in Fig.
3B. When the dose-response relationships were examined
between muscarinic agonist concentration and inhibition of
AR-stimulated AC activity, small but significant increases were seen
in the potency of muscarinic inhibition (Fig. 4). EC50s for muscarinic
inhibition of 1AR- and 2AR-stimulated activity are 0.84 ± 0.10 and 0.25 ± 0.06 µM
in young ventricles, respectively, and 0.48 ± 0.10 and 0.14 ± 0.04 µM in old ventricles. Previous studies (45) in
Fisher 344 rats also indicate that maximal muscarinic inhibition of
AR-stimulated AC activity is similar in young and old rats. Detailed
dose-response curves were not reported.
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Fig. 4.
Age increases the potency of muscarinic
inhibition of AR-mediated stimulation of
AC. Dose-response assays reveal small but significant decreases in
the EC50 for carbachol-mediated inhibition of cAMP
production stimulated by either 1ARs or
2ARs in old (open squares or
circles, respectively) versus young (filled
squares or circles, respectively) rat ventricular
membranes. 1AR stimulation was achieved with 100 µM isoproterenol + 100 µM ICI 118,551 (2AR antagonist), and 2AR
stimulation was achieved with 100 µM isoproterenol + 100 µM CGP 20712A (1AR antagonist).
n = 4 for each age group. Assays were performed in
duplicate, and representative curves are shown.
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PTX Treatment Causes a Greater Increase in AC Activity in Old
Hearts than in Young Hearts--
We further examined the relationship
between increased Gi2 levels and decreased basal and
receptor-stimulated AC activity by eliminating Gi
activity through PTX treatment. PTX ADP-ribosylates Gi/Go and prevents their interaction with
receptors, thereby removing the inhibition of AC by these G protein
subtypes (46, 47). Our previous studies have shown that PTX treatment
of human atrial membranes results in an increase in AC stimulation
through 2AR and other Gs-coupled
receptors (4), and others have shown that PTX treatment of myocytes
increases 2AR-mediated contractility (2, 6). As shown in
Table II, the inactivation of
Gi/Go by PTX increases both basal and
GPCR-stimulated AC activity, and this effect is greater in the old than
in the young left ventricular membranes for the
Gi-coupled 2ARs and glucagon receptors.
Gi inactivation in old membranes restores
receptor-stimulated cAMP production to the levels achieved in the
younger tissue. These data are consistent with our finding that more
Gi2 is activated upon stimulation of 2ARs
and other receptors in old heart (Fig. 3). 1ARs do not
couple to Gi. Accordingly, the increase in
1AR-mediated stimulation of AC following PTX treatment
was not significantly affected by age. The increase in stimulation by
forskolin, a direct activator of AC, also is not affected by age. As
expected, PTX treatment completely eliminated the inhibition of
AR-stimulated AC activity by muscarinic receptors (data not
shown).
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Table II
Effect of PTX on AC activity in young and old rat left ventricular
membranes
Receptors tested were 1ARs (measured by inclusion of 100 µM isoproterenol + 100 µM ICI 118,551 (2AR antagonist)), 2ARs (100 µM
isoproterenol + 100 µM CGP 20712A (1AR
antagonist)), and glucagon receptors (50 µM glucagon).
Direct activation of AC was tested with forskolin (50 µM). Percent Change is the increase in cAMP production
above the control level in the respective tissue in Table I.
n = 6 for each age group, and assays were performed in
duplicate. *, p < 0.05 compared with percent
change in response following PTX treatment in young membranes.
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DISCUSSION |
The main finding of the present study is that Gi2
expression in rat left ventricle increases with age. The increase in
Gi2 expression is demonstrated using several biochemical
techniques including immunoblotting, ADP-ribosylation, and
photoaffinity labeling with AA-[32P]GTP. The increase in
Gi2 results in enhanced coupling to
Gs-coupled receptors such as 2ARs and
glucagon receptors, as well as to Gi-coupled muscarinic
receptors. Thus, the net effect of the increase in Gi2
expression with age is an increase in Gi2 signaling. This results in reductions in both basal and receptor-mediated AC
activities in aged heart, both of which are restored by disabling of
Gi with PTX.
The present study examined the effect of age on cardiac
Gi, an important issue about which conflicting data
exist in the literature. Determination of the effects of age on
Gi has become increasingly important in light of the
recent demonstrations that many cardiac Gs-coupled
receptors, including 2AR, also couple to
Gi (2-4). The data in the present study clearly
indicate an increased level of Gi2 in old Fisher 344 rats, and importantly, also demonstrate an elevation in the
receptor-stimulated activation of Gi2. Prior studies in
rats had not provided a consensus as to the effects of age on cardiac
Gi. There have been reports of increased
Gi expression in Fisher 344 (21), Sprague-Dawley (19)
and Wistar rats (25), as well as an increase in Fisher 344 Gi mRNA (22). However, there also have been reports
of no change in Gi expression in Fisher 344 (22, 23) and
Wistar rats (6, 26). These differences make it necessary to examine Gi by multiple biochemical approaches. Our data
indicating Gi2 increases in immunoblotting,
ADP-ribosylation and photoaffinity labeling lead us to conclude that
there is a significant age-dependent elevation in Fisher
344 cardiac Gi2. The increase in Gi2 in aged hearts is likely not due to hypertrophy because in Fisher 344 rats
hypertrophy is seen at senescence, which occurs at 27-30 months of age
(31).
In agreement with previous reports in both human (15-17) and rat heart
(21, 23, 48-51), we also demonstrate decreases in AC activity in old
heart. To explain this phenomenon, some studies have implicated changes
at the level of AC. There has been a report of a decrease in the number
of forskolin binding sites in old rats (23), indicating a lower amount
of AC in old tissue, though there have been no reports on AC mRNA
levels in Fisher 344 rats. Although decreases in AC activity with age
could be due to decreased amounts of AC or its targets, the fact that
PTX treatment restores 2AR- and glucagon
receptor-stimulated AC signals in older hearts to the levels in younger
hearts indicates that PTX-sensitive proteins are responsible for the
decreased receptor-stimulated enzyme activity. PTX treatment in both
guinea pig heart and rat bladder results in similar reversals of
age-induced decreases in AC (20, 52). We conclude that elevated
Gi is the main cause of reduced AC activity in aged rat
left ventricles.
Apparently, an increase in cardiac Gi in aged rat heart
does not affect AR-mediated contractility. Although coupling of 1ARs to stimulation of AC and contractility through
Gs is widely accepted, questions remain as to whether
effects on contractility through 2AR involve cAMP. In
humans, stimulation of contractility via 2ARs has been
reported to occur through a cAMP-dependent mechanism that
results in protein kinase A-catalyzed phosphorylation of phospholamban,
troponin I, and C-protein, as well as enhancement of both inotropy and
lusitropy, in both non-failing (53) and failing human heart (54), as
well as in non-failing myocardium from infants with Fallot tetralogy
(55). Therefore, one would expect that in humans, an increase in the
coupling of 2AR to Gi would lower
contractility. In contrast, cAMP-independent pathways control
2AR-mediated contractility in rat (2, 56-58), cat (59), sheep (60), and dog (61). Thus, in these species, an age-induced increase in Gi would not decrease
2AR-mediated contractility. Consistent with this notion
is the finding of Jain et al. (62) who found no difference
in basal contractile and relaxation function in mice lacking either
Gi2 or Gi3.
An important functional consequence of an age-induced increase in the
coupling of cardiac 2AR to Gi2 may be
increased inhibition of apoptosis. It was shown recently that
norepinephrine, acting through a Gs pathway, increases
cardiac apoptosis (63, 64). More recent data indicate that
1ARs cause apoptosis, whereas 2ARs acting
through a Gi pathway oppose apoptosis (7-9). In adult rat ventricular myocytes, stimulation of a PTX-sensitive Gi-coupled pathway by 2AR inhibits the
number of apoptotic cells as measured by flow cytometry (7). Using a
neonatal rat myocyte model, it was shown that
2AR/Gi-mediated protection from apoptosis occurs through phosphatidylinositol 3-kinase (PI 3-kinase) and Akt/protein kinase B pathways (8). The
2AR/Gi/PI 3-kinase signaling mechanism
also has been shown to mediate the stimulation of NO production (65), a
key mechanism in the cardioprotection conferred by ischemic
preconditioning (66). Finally, 2AR- mediated protection from apoptosis recently has been reported to occur through a
Gi-dependent stimulation of p38 kinase (67),
though another study reports that 2AR activates p38
kinase through a protein kinase A-dependent pathway that
does not involve Gi (68). Thus, although the downstream
signaling molecules involved have yet to be fully elucidated, an
increase in cardiac Gi seen in aging or failing heart
may be an adaptive mechanism to protect the heart from apoptosis,
because apoptosis has been shown to occur in both aged (69, 70) and
failing heart (71, 72).
In summary, the present study provides evidence that age increases
Gi2 in older rat ventricles, and this results in more activated Gi2 upon stimulation of various GPCRs.
i2 Expression, Resulting in
Enhanced Coupling to G Protein-coupled Receptors*
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Anesthesiology, 146 Sands Bldg., Box 3094, Duke University Medical
Center, Durham, NC 27710. Tel.: 919-681-4775; Fax: 919-681-8089;
E-mail: kwatr001@mc.duke.edu.
