Nagoya J. med. Sci. 39: 1-7,1976

STUDIES ON ADENYL CYCLASE SYSTEM IN
MYOCARDIUM (PART I)
ADENYL CYCLASE SYSTEM IN THE HYPERTROPHIED
AND FAILING RABBIT HEARTSKAZUSHIGE MORI

2nd Department of Internal Medicine, Nagoya University School of Medicine(Director: Prof Tatsuo Satake)

Many reports state a marked reduction of catecholamine (CA) in cardiac tissue andan increased CA concentration of serum and urine in patients with congestive heart
failure 1)-5).
Thus, it is assumed that the function of the impared heart might besupported by the increased blood CA. It has been reported that the inotropic 6)-12) andchronotropic response 13 )-16) of CA to the cardiovascular system are mediated by adenyl
cyclase-cyclic AMP system.
Adenyl cyclase-cyclic AMP system was initially discovered as the intracellular mediatorof the glycogenolytic effects of epinephrine and glucagon in the liver by Sutherland
and Rall l ?), but it has since come to be recognized as a second messenger mediatinga variety of hormonal effects I8 )-20).
It is proposed that CA activates adenyl cyclase and increases intracellular cyclic
AMP by converting ATP to cyclic AMP which mediates inotropic action as a 2nd messenger.
Furthermore, the concentration of intracellular cyclic AMP is controlled both by adenyl
cyclase, the synthesizing enzyme, and phosphodiesterase, the. converting enzyme.
In order to investigate CA and adenyl cyclase-cyclic AMP system in cardiac failure"
the changes of myocardial CA concentration, adenyl cyclase and phosphodiesterase
activity in the hypertrophied and failing heart were investigated using experimental
aortic stenosis of rabbits.
MATERIALS AND METHODSI.

Production of cardiac hypertrophy and impending heart failure
Forty albino rabbits, weighing 2.0 - 3.0 kg, were used. They were divided into fivegroups, one group served as control. Supravalvular aortic stenosis was produced bya modification of the technique described by Tomomatsu et al 21 ). Adult rabbits wereoperated under anesthesia induced by intravenous injection of 2.5 ml/kg of urethane.
A small piece of laminallia, which was fixed at the tip of the vinyl tube (0.9 mm inside
diameter) was inserted into the right carotid artery and positioned just above the aortic
valve. After insertion, the laminallia expanded and aortic stenosis was produced. In
This study was presented at the 29th Tokai, 7th Hokuriku Regional Meeting of the Japanese
Circulation Society in Nagoya, June 27, 1970.

Received for publication December 22, 1975

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K. MORI

order to add further cardiac burden upon the hypertrophied heart, 100 ml/day ofphysiological saline solution was infused for 3 days to rabbits 30 days after the operationof aortic stenosis. These rabbits were named animals with impending heart failure.
Eight rabbits of 2 days after the operation was abbreviated by AS 2 , six of 7 days by AS?,seven of 30 days by AS 30 and nine of impending heart failure by IHF. After the animals
were sacrificed by a blow on the head, the hearts were quickly excised and the freewalls of the left ventricle were removed and used for each assay. The ratio of heart
weight to body weight was employed for the evaluation of cardiac hypertrophy.

2.

Determination of myocardial water concentration
The method described by Benson 22 ) was used for the determination of myocardial
water concentration. The water concentration was calculated from the difference between
Ig of fresh tissue of the left ventricle and dry weight obtained after drying at 110°C
for 24 hours.

3.

Determination of myocardial CA
Myocardial CA was measured spectrophotofluorometrically by the method of Von
Euler et aI 23 ) . The free walls of the left ventricle were weighed and homogenized with
5 volumes of 5% trichloro-acetic acid. The CA in the tissue extract was absorbed onto
alminium oxide and oxidized with potassium ferricyanide. The concentration of theresulting trihydroxyindole was measured in a spectrophotofluorometer.

4.

Determination of myocardial adenyl cyclase activity(a) Preparation of tissue fractions
The left ventricle was excised, minced with a fine scissor, then 0.5 gram was homogenizedin 6 volumes medium containing 0.00 I M magnesium sulfate and 0.002M glycylclycine,pH 7.5 in a homogenizer. The crude homogenate was filtered through gauze, and used
as an enzyme preparation.
(b) Determination of enzyme activities
Adenyl cyclase activity was measured by the method of Krishna et aI 24 ). The incubation
medium consisted of Tris HCl, pH 7.3, 4 x 10- 2 moles; MgCI 2 , 3.3 x 10- 3 moles; 3 H-ATP
2 x 10- 3 moles, 30 pc/p mole; theophylline, 1.0 x 10- 2 moles; epinephrine bitartrate,
1.0 x 10- 4 moles. Incubation was performed at 30°C, and was initiated by additionof 100 pi of homogenate in a total incubation volume of 0.6 m!. The reactions were
terminated at 2 minutes intervals by the 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 served for calculationof recovery, and tritiated cyclic AMP formed from the labeled ATP was separated by achromatography on Dowex-50H+ followed by barium-zinc precipitation.
Theradioactivity in an aliquot of the final supernatant fraction, representing tritiated cyclic
AMP, was counted in IS ml of a BBOT phosphor mixture in a liquid scintillation
spectrometer. The reaction was linear with myocardial homogenate over a range 0.05 ml
to 0.15 m!. Adenyl cyclase activity was expressed as mil mole of cyclic AMP formed/
h/mg of wet weight.
5.

Determination of phosphodiesterase activity
Activity of phosphodiesterase in a crude homogenate was assayed by measurement

ADENYL CYCLASE SYSTEM IN MYOCARDIUM (I)of hydrolysis of cyclic AMp 26 ).

3

The assay depends on precipitation of material withhigh absorbance at 260 mp, including products of hydrolysis, while cyclic AMP remains
in the supernatant fraction. The incubation mixture contained Tris HCI, pH 7.4, 4 x
10- 2 moles; MgClz, 2 x 10- 3 moles; and cyclic 3', 5' - AMP, 4 x 10- 4 moles in a
final volume of 1.0 m!. Hundred pI aliquot of the initial tissue homogenate was added
to initiate the reaction. Incubation was performed at 37°C and terminated by additionof 1.0 ml of 2% ZnS04 followed by 1.0 ml of 1.8% Ba(OH)2' Following centrifugation
at 4000g for 15 minutes, absorbance of the supernatant fraction was measured at 260 mp,
and the rate of degradation of cyclic AMP determined. Phophodiesterase activity was
expressed as mp mole of cyclic AMP degraded/h/mg of wet weight.
ATp· 3 H (specific activity 5 - 50 mc/m mole) was obtained from New England Nuclear
Corporation, Boston, Mass., Dowex-50H+ (SOW - X4, 200 - 400 mesh) from Bio-Rad
Laboratorus. All other compounds were obtained from commercial sources. Radioactivity
was determined with a liquid scintillation spectrometer (Aloka 100). Fluorometricmeasurement was carried out with a spectrophotofluorometer (Turner Associates).
The results were analyzed using a student's "t" test.
RESULTS

Experimental cardiac hypertrophy in rabbits
The cause of death in the operated animals was mainly attributed to heart failure
showing edematous lung, enlarged liver and ascites. The ratio of heart weight to bodyweight in 20 unoperated control animals averaged 2.39 ± 0.13 (±S.E.M.) g/kg. After theoperation, this was significantly increased above the control, and averaged 2.53 ± 0.10
g/kg in AS 2 , 2.72 ± 0.16 g/kg in AS? and 2.84 ± 0.22 g/kg in AS 30 respectively. InIHF,
the ratio, average 2.58 ± 0.09 g/kg, was slightly decreased compared with AS 3o • Myocardial
water concentration was gradually increased after the operation, averaged 778 ± 8.2 mg/g
in control animals, 786 ± 5.6 mg/g in AS 2 , 797 ± 3.2 mg/g in AS? and 802 ± 3.2 mg/g
in AS 30 respectively. In IHF, it was reduced to an average of 783 ± 14.2 mg/g. (Fig. 1)
1.

:'~K~9

HW!BW ratio*T*,,- - - - -_~%.*_._-- - ~ - ------- - .-- - - -- - -- - -----!---- --·-1
'!!'

,/"
2.0

Myocardial water concentration

mg/g

80!

,,Fig. I.

2.

~~:---,
o

2

7

--li~

,

30days~IHF

Time course changes of heart weight/body weight ratio and myocardial water concentration after aortic obstruction.
**: p<O.OI HW/BW: heart weight/body weight ratio IHF: impending heart failure

CA concentration in the left ventricle
In 34 normal rabbits, the left ventricular CA concentration averaged 1.00 ± 0.04
pg/g (±S.E.M.). In AS 2 , no significant change was observed, average 0.94 ± 0.10 pg/g.
In AS? and AS 30 , it decreased to values which averaged 0.84 ± 0.08 pg/g and 0.90 ± 0.09

4

K. MORI

pg/g respectively. IHF showed the most significant decrease in concentration, averaged
0.71 ± 0.07 pg/g (p < 0.01) compared with control. (Fig. 2)

3.

Adenyl cyclase activity in the left ventricle
In 10 nonnal rabbits, the adenyl cyclase activity in the left ventricle averaged 1.12 ±0.11 mp mole/h/mg (±S.E.M.). The activities in AS 2 and AS 7 decreased significantly,
averaging 0.69 ± 0.13mp mole/h/mg (p < 0.05) and 0.67 ± 0.14mll mole/h/mg (p < 0.05)
respectively. The values in AS 30 showed further significant decrease, averaging 0.26 ±0.12mp mole/h/mg (p < 0.01). IHF showed rather an increases ober AS 30 , averaging
0.39 ± O. 13mp mole/h/mg, which showed also a significant decrease compared withnormal. (p < 0.01). (Fig. 3)"g/g

mil mole/lvtng

..

1.0

>0

c:::
0
:;;

**

..

:;;
CJ

CG

c:::

QI

c:::

QI
III

r\l

O.

U

0

0.5

>0

U

CJ

>.
c:

-t
0

QI

"C

-t

Coot
Fig. 2.

* *

CG

~

U

1.0

0>

2 7 30dayslHF

CA concentration in the left ventricle ofrabbits in five groups.
*: p<O.OS**: p<O.OI

Fig. 3.

0Cont

2 7 30days IHF

Adenyl cyclase activity in the left
ventricle of rabbits in five groups.
*: p<O.OS**: p<O.Ol

4.

Phosphodiesterase activity in the left ventricle
The phosphodiesterase activity in the left ventricle of 10 normal rabbits averaged,
42.36 ± II.Omp mole/h/mgo The activity in AS 2 , AS 7 , and AS 30 showed no remarkablechange, averaging 36.75 ± 7.56mp mole/h/mg, 37.08 ± 5.97mp mole/h/mg and 36.2 ±6.6mp mole/h/mg respectively. The activity in IHF averaged 29.7 ± 9.8mp mole/h/mg, andalso showed no significant decrease. (Fig. 4)
DISCUSSION
The experimental method of Tomomatsu used in this study to produce cardiac
hypertrophy is simple and dependable. Cardiac hypertrophy was already observed 2 days
after the operation of aortic obstruction and progressed gradually. It is difficult in theexperimental animal to produce heart failure similar to that in humans. In this study,
impending heart failure was produced by imposing 4 days volume overload infusion ofphysiological saline solution on the animals of 30 days after the operation of aortic
obstruction. The animals with IHF showed a clinical picture which resembles patientswith congestive heart failure. In spite of some difference of seriousness in these IHF,
all cases were treated as a whole, because of difficulty of detailed classification.
In these experiments with rabbits, the left ventricular CA concentration was not
significantly depressed in 2 days after the operation of experimental cardiac failure,but was fairly depressed in 7 and 30 days, and was distinctly depressed in IHF. TheseADENYL CYCLASE SYSTEM IN MYOCARDIM (1)5results resembled that of many reporters who studied the myocardial CA in experimental
heart failure. Adenyl cyclase activity was fairly depressed in 2 and 7 days after theoperation, and further depression occured in 30 days. The degree of the depression
decreased in IHF than in 30 days without significant differences. Phosphodiesterase
activity showed a slight decrease in 2, .7, and 30 days after operation, but no significant
difference compared to normal. Many reports1).s) show decrease of myocardial CA
and increase of blood CA in cardiac failure. Increases of blood CA suggest augmented
activity of the sympathetic nervous system which is considered to serve the maintenanceof cardiac performance in heart failure. Fig. 5 shows the relation between CA andHormonem" mole/ h!rng(1 st messenger)

1

I

Itrt

0

Receptori

I

IAdenyl cyclasel,. ;·f"~I

I (1 st tarcel)

1

phosphodiesterase

ATP ~Ic-AMP1~5~AMP~f'!

+:::J~::~I

1:~j(2 nd messenger)0

response

0Fig. 4.

"--

Cont

-r

Effector cell,;A '<,2 7 30days IHF

Phosphodiesterase activity in the left
ventricle of rabbits in five groups.

Fig. 5.

The second messenger system (fromSutherland et.a1. 2S»).

adenyl cyclase-cyclic AMP system 2S). Myocardial cyclic AMP which play an importantrole as a second messenger in the inotropic effect of myocardial contraction is controlled
by adenyl cyclase and phosphodiesterase. There are few reports on myocardial adenyl
cyclase activity in cardiac hypertrophy and heart failure. On one hand, Sobel et al~{;).
have reported a depressed adenyl cyclase activity in failing guinea pig heart, while on
the other hand, Gertler, Saluste, and Spencer27 ) failed to observe such a change.
In this study, in spite of no changes observed on converting enzyme phosphodiesterase
in cardiac hypertrophy and IHF, significant decreases of synthesizing enzyme adenyl
cyclase activity suggest an important defect of cyclic AMP producing system in cardiac
hypertrophy and IHF. However a direct measurement of my ocardial cyclic AMP would
be in cardiac hypertrophy and cardiac failure. The mechanism of depletion of myocardialadenyl cyclase activity in cardiac hypertrophy and heart failure is not clear at present. The
enzyme dilution theory due to increased myocardial protein in heart failure by Sobel et
a1. 26 ) cannot be neglected, but all can not be explained by it, because there was no depression of myocardial phosphodiesterase activity. Furthermore, though the depletion of
myocardial CA was noticed in cardiac hypertrophy and impending heart failure, it seemedthat the depressed myocardial adenyl cyclase activity in this stage was not due to decreaseof myocardial CA store. It is supported by the result of Sobel's experiment using a cat thatno diminntion of adenyl cyclase activity was observed when myocardial CA was reduced bydenervation. From the above reasons adenyl cyclase itself seemed to receive primary qualitative change in cardiac hypertrophy and heart failure. In any case, prominent depression of

6

K. MORI

myocardial adenyl cyclase activity in cardiac hypertrophy and heart failure indicates disturbance of myocardial cyclic AMP producing system which play an important role on
inotropic action in heart failure.
Two biochemical defects in myocardial cyclic AMP producing system, the decreaseof myocardial CA and the depression of adenyl cyclase activity were demonstrated in
cardiac hypertrophy and heart failure and it is suggested that the diminished myocardial
inotropic effects would be explained by them.
SUMMARY
In order to investigate the pathophysiological significance of myocardial CA and adenyl
cyclase-cyclic AMP system in cardiac failure, myocardial CA, adenyl cyclase andphosphodiesterase activity in cardiac hypertrophy and impending heart failure were
measured by experimental aortic stenosis in rabbits.
I.
A reduction of left ventricular CA concentration was observed in the stage of
cardiac hypertrophy and significant reduction in the stage of impending heart failure.
2. Adenyl cyclase activity decreased gradually after the production of experimental
hypertrophy and most significantly in 30 days after and the stage of impending heart
failure.
3. Phosphodiesterase activity showed no significant changes in both the stage of
cardiac hypertrophy and impending heart failure.
These results suggest that at least two biochemical defects in the supporting mechanismof sympathetic systems in cardiac failure - the depletion of myocardial CA and thedecrease of adenyl cyclase activity - may contribute to the redu~tion of myocardial
inotropic effect.
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