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Depressed Adenyl Cyclase Activity
in the Failing Guinea Pig Heart
By Burton E. Sobel, M.D., Philip D. Henry, M.D.,
Alice Robison, A.B., Colin Bloor, M.D., and John Ross, Jr., M.D.
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ABSTRACT
There is considerable evidence that several effects of catecholamines on beta
receptors, including the positive inotropic effect on the heart, are mediated by
cyclic-AMP. Myocardial cyclic-AMP concentration depends on the activity of
at least two enzymes: adenyl cyclase, regulating synthesis, and phosphodiesterase, regulating degradation of the compound. In view of the importance
of adrenergic support in maintaining cardiac performance, we examined synthesis and degradation of cyclic-AMP in homogenates of hearts from guinea
pigs with congestive heart failure produced by constriction of the ascending
aorta and from sham-operated controls. In the experimental group, left ventricular weight, DNA, RNA, and protein content were significantly increased
7 to 12 days following operation, and congestive heart failure developed,
manifested by hydrothorax, hepatomegaly, and histologic evidence of pulmonary edema and passive congestion of viscera. Adenyl cyclase activity in
homogenates from the experimental group was 36% less than that observed in
controls ( P < 0.001). However, phosphodiesterase activity was unchanged.
These findings suggest that there is a double biochemical defect in the adrenergic support to the failing heart. In addition to the known reduction of
myocardial catecholamines, there appears to be diminished capacity for synthesis
of cyclic-AMP, an important mediator of catecholamine action.
ADDITIONAL KEY WORDS
catecholamines
myocardial contractility
cyclic-AMP
experimental heart failure
effects on beta receptors
• There is considerable evidence suggesting
that the positive inotropic action of catecholamines provides an important reserve mechanism in the regulation of cardiac performance
(1, 2). It is clear that effects of catecholamines on beta receptors, such as glycogenolysis in the heart and lipolysis in adipose tissue,
are mediated by a "secondary intracellular messenger," 3',5'-adenosinemonophosphate
(cyclic-AMP) (3). Furthermore, it appears
that the positive inotropic effect of catecholamines on the heart, another effect on beta
receptors, may be mediated by cyclic-AMP (4,
5), although this has not been established
From the Departments of Medicine and Pathology,
University of California at San Diego, La Jolla,
California 92037.
This work was supported in part by U. S. Public
Health Service Grant HE 12373 from the National
Heart Institute.
Received October 29, 1968. Accepted for publication February 19, 1969.
Circulation Research, Vol. XXIV, April
1969
unequivocally (6). Intracellular concentration
of cyclic-AMP is regulated by the activity of at
least two enzymes: adenyl cyclase, regulating
synthesis, and phosphodiesterase, regulating
degradation. Adenyl cyclase can be activated
by several agents, including catecholamines,
in vitro and in vivo. Fluoride ion produces
maximal activation of the enzyme in vitro
(3).
Recently, it has been shown that myocardial
catecholamine stores are depleted during both
spontaneous heart failure (1) and that produced experimentally (7), although circulating levels of catecholamines are elevated.
Since reduction of adenyl cyclase activity
would compound the physiologic effects of
catecholamine depletion, the present study
was undertaken to examine synthesis and
degradation of cyclic-AMP in the guinea pig
heart overloaded by aortic constriction. Determinations of enzymes involved in the
507
508
SOBEL, HENRY, ROBISON, BLOOR, ROSS
synthesis and degradation of cyclic-AMP were
performed using homogenates from failing left
ventricular myocardium and corresponding
tissue from sham-operated controls. Activity of
adenyl cyclase in homogenates from the
failing heart was significantly reduced. However, there was no concomitant reduction of
phosphodiesterase activity. These studies suggest that loss of intrinsic adrenergic support to
the failing myocardium may be explained not
only by myocardial catecholamine depletion
but also by reduced capacity to generate
cyclic-AMP in response to catecholamines.
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Materials and Methods
Chemical Compounds.—Reagents of the highest
grade available commercially were prepared in
deionized and charcoal-filtered water. Tritiated
adenosine-5'-triphosphate, tetralithium salt (ATP)
was obtained from Schwarz BioResearch; 3',5'adenosinemonophosphate (cyclic-AMP) and carrier dipotassium ATP from Sigma; Dowex-50 H-f(Bio Rad AG-50W X-4, 200-400 mesh) and crystalline bovine serum albumin from Calbiochem;
theophylline and epinephrine bitartrate from K & K
Laboratories; and 2,5-bis-[2-(5-tert-butylbenzoxazolyl) ]-thiophene (BBOT) from Packard.
Animals.—Male guinea pigs weighing between
400 and 500 g were anesthetized with sodium
pentobarbital, 30 mg/kg ip. Positive-pressure
respiration was maintained with a face mask, as
described by Loring (8). The chest was entered
through the third intercostal space and the
ascending aorta exposed transpericardially. In the
experimental group, aortic constriction was produced by applying a circumferential teflon clip of
2.1-mm internal 'diameter proximal to the origin
of the brachiocephalic vessels (9, 10). Clips of
this size led to the development of ventricular
hypertrophy and congestive heart failure as
evidenced by increased heart weight, total left
ventricular deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein; hydrothorax;
and histologic evidence of pulmonary edema and
passive congestion in the lung, kidney, liver, and
spleen. In the control group, sham operations
were performed. Following pericardiectomy, the
ascending aorta was isolated but placement of the
clip was omitted. In two control animals, a
nonconstricting clip was used with no change in
results.
Histologic Studies.—Since use of tissue for
enzyme determinations precluded microscopic
analysis of the heart in some animals in the
experimental group, identically operated weightmatched controls were used for histologic study.
These were killed at corresponding times postoperatively and routine sections of heart and viscera
obtained. Gross findings in animals used for
enzyme studies were the same as those in animals
studied histologically, and in all cases included
cardiac hypertrophy, hydrothorax, and hepatomegaly.
Preparation of Tissue Fractions.—All preparative procedures were performed at 0 to 4°C.
Seven to ten days postoperatively control and
experimental animals were stunned, and the heart
was rapidly removed, blotted, immersed in 0.25M
sucrose, and weighed. The left ventricle was excised, minced with a fine scissors, and passed
through a tissue press with holes 1 mm in diameter.
It was then homogenized in 15 volumes per gram
of mince in a medium containing 0 . 0 0 1 M magnesium sulfate and 0.002M glycylglycine, pH 7.5, in
a VirTis "45" homogenizer with a speed setting of
5 for two 15-second bursts, and subsequently by
hand in a Dounce homogenizer. The crude
homogenate was filtered through two layers of 60mesh cheesecloth. Aliquots from control and
experimental tissue were used for phosphodiesterase determinations. The particulate fraction was
obtained by centrifugation of the crude homogenate at 2,000g for 15 minutes, followed by two
washes of sedimented material suspended in 10
ml of homogenizing medium and centrifuged at
3,700g for 15 minutes. The final pellet was
suspended in homogenizing medium, and aliquots
were used for determination of adenyl cyclase
activity.
Biochemical Determinations.—Left ventricular
dry weight was determined following lyophilization for 48 hours by which time weight remained
constant. Protein concentration in the crude
homogenate and the particulate fraction was
determined by the standard Lowry procedure
(11); fractions were adjusted such that the
control and experimental fractions had the same
concentration prior to enzyme assay. Adeny]
cyclase activity was measured by the method of
Krishna et al. (12) and Brodie et al. (13). The
incubation medium consisted of Tris HC1, pH 7.3,
4 X 10- 2 M; MgCl2, 3.3 X 1 ( H M ; 3H-ATP, 2.0 X
3
2
10- M, 30 /xc/fimo\e; theophylline, 1.0 X 10" M.
When sodium fluoride or epinephrine bitartrate
were added as activators, they were present in
concentrations of 1.0 X 10~ 2 M and 1.0 X I O ^ M ,
respectively. Incubations were performed at
30°C, and were initiated by addition of 100
^/.liters of tissue suspension (0.8 mg to 1.1 mg
protein) in a final volume of 0.6 ml. The reactions
were terminated at 2-minute intervals by addition
of 0.5 mg of carrier cyclic-AMP and immediate
immersion in a boiling water bath for 3 minutes.
Incubations employing heat-denatured enzyme
were run as blanks. The carrier cyclic-AMP
Circulation Research, Vol. XXIV, April 1969
509
ADENYL CYCLASE IN HEART FAILURE
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served for calculation of recovery, and tritiated
cyclic-AMP formed from the labeled ATP was
separated by chromatography on Dowex-50 H +
followed by barium-zinc precipitation as described previously (12, 14). Radioacitvity in an
aliquot of the final supernatant fraction, representing tritiated cyclic-AMP, was counted in 15
ml of a BBOT phosphor mixture in a Packard
liquid scintillation spectrometer. Another aliquot
was used to calculate recovery of added cyclicAMP by determination of absorbance at 260 m/x
using a molar absorption coefficient (AM26O) of
14.3 X 103.
Activity of phosphodiesterase in the crude
homogenate was assayed by measurement of
hydrolysis of cyclic-AMP. The assay depends on
precipitation of material with high absorbance at
260 m/ji, including products of hydrolysis, while
cyclic-AMP remains in the supernatant fraction.
The incubation mixture contained Tris HC1, pH
7.4, 4 X 10- 2 M; MgClo, 2 X 10- 3 M; and cyclic 3',
5'-AMP, 4 X I O ^ M in a final volume of 1 ml. A
100-|Ltliter aliquot of the initial tissue homogenate
(0.6 mg protein) was added to initiate the reaction. Incubations were performed at 37°C and
terminated by addition of 1 ml of 2% ZnSO4 followed by 1 ml of 1.8% Ba(OH) 2 . Following centrifugation at 4000g for 15 minutes, absorbance of
the supernatant fraction was measured at 260 m/j,,
and the rate of degradation of cyclic-AMP determined.
DNA and RNA were separated by the modified
Schmidt-Thannhauser procedure (15) and assayed by the diphenylamine reaction (16) and
ultraviolet absorption using a double-wavelength
method to correct for peptide fragments (17).
Results
The evidence that the experimental procedure produced cardiac hypertrophy and failure
is summarized in Table 1. These data were
obtained from experimental animals subjected
to the identical surgical procedure and killed
at corresponding times. In 13 guinea pigs
killed 5 to 10 days after operation, postmortem examination consistently showed bilateral pleural and pericardial effusions,
edematous lungs, and congested viscera.
Ventricular weight and heart weight-body
weight ratio were significantly greater in
experimental animals than in controls (Table
1). In addition, left ventricular DNA, RNA,
and protein content were elevated in the
experimental group, findings in agreement
with previous reports (18-20). Histological
sections of the hearts revealed myocardial
edema, hypertrophied myocardial fibers, and
diffuse multiple foci of myocardial necrosis.
Microscopic examination of the lungs, liver,
spleen, kidney, and adrenals of experimental
guinea pigs confirmed the presence of pulmonary congestion and edema and passive
congestion of the viscera. These post-mortem
findings substantiated the fact that in the
experimental group the hearts were significantly hypertrophied and that congestive
heart failure was present.
The activity of adenyl cyclase in particulate
fractions from the failing guinea pig heart was
markedly reduced when compared with controls (Table 2). It is important to note that
the percent of homogenate protein appearing
in the 2000g pellet was the same in control
(46%, N = 6) and in experimental (45%,
N = 5) preparations. Furthermore, the percent of adenyl cyclase activity in the homogenate obtained in the 2000g pellet was also
similar (control = 68%; experimental = I
TABLE 1
Alterations in Guinea Pig Myocardium Following Aortic Constriction
Heart weight*
body weight
Control
(N = 4)
Aortic constriction
(N = 4)
LV dry weight
LV wet weight
LV content (mg/kg body weight)
DNA
RNA
Protein
0.239 ± 0.006
23.0 ± 0.9
2.88 ± 0.15
4.76 ± 0.32
495 ± 45
0.468 ± 0.056t
21.1 ±0.9
5.85 ± 0.46t
9.38 ± l.Olf
937 ± 70f
"Combined right ventricular and left ventricular weight. LV = left ventricular. Values are means ± SEM. Significance of the differences between the control and experimental values was tested using Student's t-test. fDifferences
were significant at the 0.01 level.
Circulation Research, Vol. XXIV, April 1969
510
SOBEL, HENRY, ROBISON, BLOOR, ROSS
TABLE 2
Adenyl Cyclase Activity in Control and Failing Guinea Pig Myocardium
Particles from
control hearts*
160
200
198
155
189
162
196
214
Expt.
1
2
3
4
5
6
7
8
MEAN I t SE
Whole
homogenate
Particles from control
hearts — particles from
Particles from
Whole
failing hearts
failing hearts homogenate
77
105
139
184 ± 7.8f
133
83
87
150
48
103
131
103
124
119
52
58
59
61
73
72
95
118 ± 8.0f
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Results are piconioles of cyclic-AMP produced/ing protein/min and represent averages of three determinations
of initial reaction rate in incubation media containing sodium fluoride, 1 X 10~ 2 M as an activator and 0.8 to 1.1 mg
enzyme protein. Typical kinetics exhibited by the pellets are illustrated in Figure 1. Whole homogenates showed
linear reaction rates for 4 minutes in each experiment. *2000g washed pellet. fThe significance of the differences
was tested using Student's t-test. P < 0.001.
N = 4). Representative experiments are illustrated in Figure 1. In each experiment, several
incubations were performed and all preparations exhibited typical kinetics. The maximal
activity, obtained with fluoride in the incubation medium, was consistently lower in the
experimental preparations. In additional experiments, activation by epinephrine was
examined. Control and experimental preparations demonstrated similar activation (39% of
maximal activity [N = 4] and 34% [N = 4],
respectively), indicating that there was no
specific impairment to activation of adenyl
cyclase by catecholamines. Both preparations
demonstrated approximately 20% of maximal
activity in the absence of any added activator.
The specific activity of myocardial adenyl
cyclase observed in this study was greater
than that found previously in dog myocardium (3). For this reason, radiochemical purity
of the precursor 3H-ATP was verified chromatographically, and internal standards were
used in the counting procedure. The high
enzyme activity observed in the present study
may reflect species difference, since values
obtained with cat myocardium were appreciably lower (14).
The activity of phosphodiesterase in homogenates from the failing heart was essentially identical to that in control tissue (Table
2
4
TIME (minutes)
6
FIGURE 1
Accumulation of cyclic-AMP by homogenates from
failing and control myocardium. Incubation media
included 1.0 X 10~SM sodium fluoride as an activator
of adenyl cyclase and 0.8 mg of of enzyme protein
(see text). Data from representative experiments on
one control guinea pig and one with heart failure.
Circulation Research, Vol. XXIV, April 1969
511
ADENYL CYCLASE I N HEART FAILURE
TABLE 3
Phosphodiesterase Activity in Control and Failing Guinea Pig Myocardium
m/i moles cyclic-AMP hydrolyzed/mg protein/15 minutes
Control
Failing
hearts
hearts
Expt.
1
2
3
4
5
MEAN ± SE
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3). Reaction rates were linear for 15 minutes
and proportional to enzyme concentration.
The fact that activity of phosphodiesterase per
milligram of protein was not diminished in the
failing heart suggests that there is selective
alteration of enzyme activities favoring diminished synthesis without concomitantly reduced degradation of cyclic-AMP in myocardium exposed to outflow tract obstruction.
Discussion
The major finding of this study, namely that
adenyl cyclase activity is diminished in the
experimentally failing heart, has several implications. It indicates that there is an additional
biochemical alteration which may impair
adrenergic function in experimental heart
failure. On the one hand, myocardial catecholamine stores are diminished, and hence
sympathetic stimulation is less effective in
producing a positive inotropic response (21).
On the other hand, whatever endogenous
catecholamines are released may be less
effective in generating intracellular cyclicAMP because of the reduced adenyl cyclase
activity. One would anticipate that an impaired positive inotropic response might occur
even in the presence of elevated circulating
catecholamines if cyclic-AMP is the mediator
of this effect. Since the failing heart manifests
normal phosphodiesterase activity, indicating
an undiminished capacity to degrade cyclicAMP, it seems possible that the failing heart
might be unable to maintain physiologically
significant intracellular levels of cyclic-AMP
even transiently. However, definitive information regarding this point must await further
studies on myocardial levels of cyclic-AMP.
Circulation Research, Vol. XXIV, April 1969
94
119
142
99
139
119 ± 10
115
114
139
91
134
119 ± 9
The mechanism of reduction of myocardial
adenyl cyclase activity in experimental heart
failure is not yet clear. The reduction in
activity may reflect dilution of enzyme activity
because of a generalized increase in myocardial protein. However, since the phosphodiesterase activity is not diminished, dilution
appears to be nonuniform with respect to
enzymes regulating cyclic-AMP synthesis and
degradation. Thus, failing myocardium appears to manifest a reduced capacity for
synthesis of cyclic-AMP without a comparably
reduced capacity for degradation. Diminution
of adenyl cyclase activity probably does not
result from reduced catecholamine stores per
se, since depletion of myocardial catecholamines consequent to extrinsic denervation
does not lead to reduction of adenyl cyclase
activity in experimental animals (14).
There is increasing evidence that the
inotropic and metabolic effects of cyclic-AMP
may be independent, although both may
reflect activity of the same secondary messenger (4, 6). Thus, the presence or absence
of metabolic manifestations reflecting altered
adenyl cyclase activity does not necessarily
establish the presence or absence of important
reduction of the catecholamine-induced positive inotropic effect.
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Circulation Research, Vol. XXIV, April 1969
Depressed Adenyl Cyclase Activity in the Failing Guinea Pig Heart
BURTON E. SOBEL, PHILIP D. HENRY, ALICE ROBISON, COLIN BLOOR and JOHN
ROSS, Jr.
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Circ Res. 1969;24:507-512
doi: 10.1161/01.RES.24.4.507
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