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A Possible Change in the Rate-Limiting Step for
Cardiac Norepinephrine Synthesis in the
Cardiomyopathic Syrian Hamster
M I C H A E L J. S O L E , A R V I N D B . K A M B L E , A N D M .
NASIR
HUSSAIN
SUMMARY The development of heart failure in the cardiomyopathic hamster is associated with a decrease in
norepinephrine stores and parallel increases in cardiac sympathetic tone and tyrosine hydroxylase activity. Despite
the increase in tyrosine hydroxylase, cardiac norepinephrine synthesis does not increase in heart failure. In this
study, we have shown that an accumulation of cardiac dopamine accompanies the decline of cardiac norepinephrine.
The abnormal content of norepinephrine and of dopamine in the decompensating hamster heart is restored to
normal by peripheral ganglionic blockade. The acute increase in cardiac sympathetic tone induced by immobilization
stress in control hamsters mimics the alterations in cardiac catecholamine distribution found in heart failure. Other
investigators have demonstrated similar alterations in the catecholamine content of the rat submaxillary gland and
adrenal medulla following an increase in sympathetic input to these organs. We conclude that the increase in cardiac
sympathetic tone in the late stages of hamster cardiomyopathy appears to lead to a shift in the rate-limiting step for
norepinephrine synthesis from the hydroxylation of tyrosine to the hydroxylation of dopamine. There is evidence
that this shift which results in an accumulation of dopamine in the noradrenergic nerve terminals of the heart is a
general manifestation of augmented sympathetic nerve traffic rather than a peculiarity of hamster cardiomyopathy.
T H E C A R D I O M Y O P A T H I C hamster is a reproducible
spontaneous model of chronic congestive heart failure
and thus may be a useful paradigm for human myocardial
disease. 1 The development of heart failure in hamster
cardiomyopathy is associated with a decrease in cardiac
norepinephrine stores 2 - 3 and an increase in the rate constant for cardiac norepinephrine turnover, a neurochemical index of cardiac sympathetic tone. 2
From the Department of Medicine, University of Toronto, Toronto,
Ontario, Canada.
Supported by the Ontario Heart Foundation (1-42) and by Grant HL
18824-01 from the U.S. Public Health Service.
Dr. Sole is a Senior Fellow of the Ontario Heart Foundation.
Address for reprints: Dr. Michael J. Sole, Clinical Sciences Division,
Medical Sciences Building, University of Toronto, Toronto, Ontario,
Canada M5S 1A8.
Received November 10, 1976; accepted for publication May 18, 1977.
The putative rate-limiting step for the biosynthesis of
norepinephrine is the hydroxylation of tyrosine to dopa
catalyzed by the enzyme tyrosine hydroxylase.4 We observed an increase in the activity of this enzyme in the
myopathic hamster heart during cardiac decompensation.5
This increase in tyrosine hydroxylase correlated well with
the increase in cardiac sympathetic tone. However, cardiac norepinephrine synthesis remained relatively constant, failing to reflect these increases. It is possible that,
despite the measured increase in tyrosine hydroxylase
activity in vitro, changes in precursor, product, and cofactor relationships obtaining in vivo obviated an increase in
in vivo synthesis. It also was possible that the hydroxylation of tyrosine was no longer the rate-limiting step for
norepinephrine synthesis in the failing myopathic hamster
heart and that the hydroxylation of dopamine became
NOREPINEPHRINE SYNTHESIS IN HAMSTER CARDIOMYOPATHY/So/e et al.
rate-limiting. If the latter were true, we would expect to
find an accumulation of cardiac dopamine accompanying
the decline of norepinephrine stores. To evaluate this
latter hypothesis, we examined the cardiac contents of
dopamine and norepinephrine in cardiomyopathic and
control hamsters at rest and during alterations in cardiac
sympathetic tone.
Methods
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Both norepinephrine and dopamine were measured in
the hearts of cardiomyopathic hamsters (Bio 53.58;
TELACO) and of their age- and sex-matched controls
(RB) at representative stages in the course of the cardiomyopathy. The hamsters were killed by decapitation.
Their hearts and thigh muscles were removed and rinsed;
the ventricles were dissected free of the atria and great
vessels. The tissues immediately were frozen on dry ice.
The muscle specimens and ventricles were weighed, homogenized with a Polytron homogenizer in 30 volumes of
iced 0.1 N perchloric acid, and centrifuged at 30,000 g
for 30 minutes. The supernatant fluids were stored at
— 75°C before analysis.
Norepinephrine and dopamine were assayed by a micromodification of the radioenzymatic method of Coyle and
Henry6 exactly as described by Palkovits et al.7 In this
assay, dopamine and norepinephrine in the tissue supernatant fluids were converted to their O-methylated analogues in the presence of catechol-0-methyl transferase
and S-adenosyl-methionine(3H-methyl) (specific activity,
11.6 Ci/mmole; New England Nuclear). The labeled
normetanephrine and 3-methoxytyramine were extracted
and the former was converted to vanillin(3H-methyl) by
metaperiodate cleavage. The methoxytyramine(3H-methyl)
and vanillin(3H-methyl) were separated by solvent extraction and counted in a liquid scintillation counter.
To calculate norepinephrine turnover in skeletal muscle, a-methyl-para-tyrosine (250 mg/kg), an inhibitor of
norepinephrine synthesis, was administered by intraperitoneal injection at intervals of 3 hours. Three groups of
five 240- to 270-day old hamsters from each of the
myopathic and control strains were killed at 0, 3, and 6
hours. The turnover rate constant was calculated from
the rate of decline of the logarithm of the muscle norepinephrine concentration (regression coefficient). Analysis
of variance was used for calculating the standard error of
the regression coefficient and the significance of the
regression coefficients.
Chlorisondamine (Ecolid chloride), a ganglionic blocker
which does not enter the central nervous system, was
used to inhibit peripheral sympathetic activity in one of
our studies; 10 mg/kg was given to the treated hamsters
by intraperitoneal injection every 6 hours for 24 hours.
Untreated hamsters received the 0.9% saline vehicle.
The hamsters were killed at 24 hours and the hearts were
taken for catecholamine analysis.
For some experiments, hamsters were stressed by immobilization2 by taping their limbs to a board. Their
heads were free to move and they were allowed access to
water, but not food. The animals were left undisturbed
and not in pain. At 24 hours, the hamsters were decapitated while still immobilized. The experiment adhered to
815
TABLE 1 Norepinephrine and Dopamine Levels in
Hamster Hearts
Norepinephrine
concentration
Dopamine concentration
1245 ± 50
1306 ± 121
1231 ± 117
1288 ± 8 7
1699 ± 147
849 ± 92*
112 ± 11
105 ± 17
127 ± 17
173 ± 15
109 ± 8
1577 ± 141*
(ng/g)
Control (30-40 days old)
Myopathic
Control (140-160 days old)
Myopathic
Control (240-270 days old)
Myopathic
(ng/g)
Each value is the mean ± SE for 7-9 hamsters.
* Differs from control at P < 0.001.
the rules for the humane treatment of animals as set by
the University of Toronto.
Results
The cardiomyopathy of the Bio 53.58 hamster, like
that of other cardiomyopathic hamster strains, may be
divided into several pathophysiological stages.1 At 30-40
days of age, areas of focal myolysis and cellular infiltrates
appear in the hearts of myopathic animals. Although
these lesions appear to "heal" in the following 2 months,
the myocardial mass gradually increases and the heart
dilates. The terminal stages of the heart disease in the
myopathic hamsters used in these experiments occurred
at 240-270 days of age. Preliminary experiments demonstrated that cardiac sympathetic tone and tyrosine hydroxylase activity greatly increased during cardiac decompensation in a manner similar to that previously reported for
the Bio 14.6 strain.2-5
Dopamine levels were approximately 8-9% of norepinephrine levels in the hearts of hamsters 30-40 days old,
and 10-13% of norepinephrine levels in those 140-160
days old (Table 1). There was no difference in cardiac
catecholamine stores between normal and myopathic
hamsters during these stages. We found a striking change
in cardiac norepinephrine and dopamine levels in 240- to
270-day-oId myopathic animals (Table 1). The concentration (and content) of cardiac norepinephrine was a fraction of that found in the controls. Cardiac dopamine, on
the other hand, was increased several-fold in an almost
stoichiometric fashion. It was possible that our observations reflected a neurohumoral abnormality found generally in hamster dystrophic muscle. Therefore, we measured norepinephrine and dopamine in the.hindleg muscles of failing dystrophic and control hamsters (Table 2).
TABLE 2 Catecholamine Levels and Turnover Rate in
Skeletal Muscle of Myopathic and Control Hamsters
Control
NE/g muscle (ng/g)
DA/g muscle (ng/g)
NE turnover rate
stant (hours"1)
NE half-life (hours)
54.7 ± 3.9*
16.6 ± 2.0
0.192 ± 0.03
3.6
Myopathic
66.5 ± 3.8
10.0± 1.1 +
0.209 ± 0.03
3.3
NE = norepinephrine; DA = dopamine; half-life = 0.693/
rate constant.
• Mean ± SE for seven hamsters.
t Differs from control at P < 0.025.
CIRCULATION RESEARCH
816
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We found no differences in either norepinephrine concentration or turnover. Dopamine concentration in myopathic
muscles was reduced. We previously had shown that
cardiac norepinephrine stores in failing hamsters of the
14.6 strain could be restored following peripheral ganglionic blockade by chlorisondamine.2 Chlorisondamine administration to control hamsters had no effect on either
the distribution or the steady state levels of cardiac
catecholamines (Fig. 1). Prior to chlorisondamine treatment, the content of cardiac norepinephrine and dopamine in terminally ill myopathic hamsters was approximately 12% and 550% of control values, respectively.
Dopamine content was 7 times that of norepinephrine in
myopathic hearts. Both the dramatic decrease in norepinephrine and increase in dopamine in failing myopathic
hearts were completely abolished by peripheral ganglionic
blockade (Fig. 1).
It appeared that alterations of sympathetic tone were
capable of affecting catecholamine distribution within the
failing heart. We wished to determine whether these
observations held true for a relatively acute increase in
sympathetic tone in normal hearts. We stressed control
hamsters by immobilization2 for 24 hours. Cardiac norepinephrine levels fell (Fig. 2); cardiac dopamine, however,
showed a significant and almost stoichiometric rise.
Discussion
Congestive heart failure in the cardiomyopathic hamster
is accompanied by an increase in cardiac sympathetic
tone and a decrease in cardiac norepinephrine stores.2
We have demonstrated that an accumulation of dopamine
accompanies the decline of norepinephrine in the decompensating hamster heart. The abnormal contents of both
norepinephrine and dopamine could be restored completely to normal values by peripheral ganglionic blockade. Thus, alterations in sympathetic tone dramatically
affected catecholamine distribution within the sympathetic
nerve endings of the failing heart.
A substance with catecholamine-like histofluorescent
properties has been described as accumulating in the
[ i Dopamine
^ B Norepinephrine
600
400
200
Untreated
Treated
Control
Untreated
Treated
Myopathic
FIGURE 1 The effect of chlorisondamine on the cardiac catecholamine content of hamsters 240-2 70 days old. Each value is the
mean ± SE for 6-8 hamsters. * = P < 0.001 for myopathic
untreated vs. control untreated; ** = P < 0.001 for myopathic
treated vs. untreated.
VOL. 41, No. 6, DECEMBER
1977
EHI Rest
700
I Stress
600
500
£
o
o
400
CD
I
300
Io
200
100
Norepinephrine
Dopamine
FIGURE 2 The effect of immobilization stress on the cardiac
catecholamine content of control hamsters 240-270 days old.
Each value is the mean ± SE for 8-9 hamsters. * = P < 0.01 for
stressed vs. unstressed; " = P < 0.005 for stressed vs. unstressed.
skeletal muscle fibers of patients with sex-linked pseudohypertrophic muscular dystrophy.8 The noradrenergic innervation of the dystrophic hamster heart has been examined by the formaldehyde fluorescent histochemical technique of Falck and Hillarp.3 Definite alterations in both
the configuration and intensity of catecholamine fluorescence were seen in the nerve terminals of dystrophic
hearts, but there was apparently no accumulation of
intrafibrillar monoamines. Gordon and Dowben9 found a
decrease in cardiac norepinephrine and an increase in
skeletal muscle norepinephrine and epinephrine in the
dystrophic mouse. Although they did not measure tissue
dopamine, they did note elevations in the urinary excretion of dopamine as well as of norepinephrine and epinephrine. A significant increase in the urinary excretion
of norepinephrine and epinephrine by myopathic hamsters
was found by Kabara et al.10 However, these workers
failed to find a change in urinary dopamine. We found no
increase in either the norepinephrine turnover rate or the
dopamine concentration of dystrophic hamster skeletal
muscle. Thus the increase in both norepinephrine turnover
and dopamine stores of the heart was not a manifestation
of a neurohumoral abnormality found generally in the
dystrophic striated muscles of the hamster.
The rates of individual steps in a sequence of enzymatic
reactions are determined by the steady state concentration
of each intermediate in the sequence as well as the kinetic
characteristics and concentration of each enzyme. The
rate of norepinephrine synthesis in the decompensating
hamster heart remains relatively constant in spite of an
increase in both cardiac sympathetic tone and tyrosine
hydroxylase activity. In this setting, the accumulation of
dopamine in the presence of a decrease in norepinephrine
is compatible with a shift in the rate-limiting step for
norepinephrine synthesis from the hydroxylation of tyrosine to the hydroxylation of dopamine.
NOREPINEPHRINE SYNTHESIS IN HAMSTER CARDIOMYOPATHY/So/e et al.
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The conversion of dopamine to norepinephrine is complex. Dopamine-/3-hydroxylase, like tyrosine hydroxylase,
may be induced by an increase in sympathetic tone."
Dopamine-/3-hydroxylase, however, is located largely
within the noradrenergic nerve granules; thus, in response
to sympathetic stimulation, the enzyme is lost from the
nerve terminal by exocytosis.12-13 An imbalance between
dopamine-/3-hydroxylase production and loss could cause
enzyme activity to fall13 to the point of being inadequate
to meet heightened demands for norepinephrine synthesis.
Furthermore, as dopamine-/3-hydroxylase is sequestered
within the noradrenergic nerve granule, dopamine must
be transported across the vesicular membrane prior to
hydroxylation. An imbalance between the production and
loss of nerve granules could theoretically reduce the
number of dopamine transport and/or norepinephrine
storage sites and thus limit dopamine conversion. The
inherent kinetic characteristics of the dopamine transport
process itself could also be limiting. Finally, dopamine-/3hydroxylase appears to be regulated both by cofactors14
and endogenous inhibitors.15 It is possible that the relationship of the enzyme to these modifiers could change in
response to alterations in sympathetic nerve traffic.
These observations also appear to hold for relatively
acute increases in sympathetic tone in normal hamsters.
The severe stress imposed on control hamsters by prolonged immobilization resulted in a decrease in cardiac
norepinephrine and an increase in cardiac dopamine similar to that found in hamster heart failure. Other studies
also support the possible general applicability of our
observations. An increase in adrenal medullary dopamine
has been described following neurogenic stimulation of
the adrenal.1" In addition, Snider et al.17 showed that
electrical stimulation of the sympathetic nerves supplying
the submaxillary gland of the rat leads to both a decrease
in norepinephrine and an increase in dopamine in the
stimulated gland. Their results indicated that this dopamine was bound largely to granules in the nerve terminal.
These results are particularly interesting in the light of
recent studies examining negative feedback mechanisms
for neurotransmitter release. Dopamine appears to be a
potent inhibitor of norepinephrine release from peripheral
sympathetic nerve endings.18"20 There is some evidence
that this inhibition may be mediated by presynaptic dopaminergic receptors.18"20 If dopamine, in response to an
increase in sympathetic tone, were to accumulate in a
functionally releasable pool, one might postulate that it
acts to inhibit excessive norepinephrine release and thus
conserve neurotransmitter stores.
Our experiments provide no indication of whether
cardiac dopamine in the stressed or failing hamster is
largely in a functionally releasable pool or indeed is intraor extravesicular. Further experiments examining the conversion of tyrosine into dopamine and dopamine into
norepinephrine under conditions of increased sympathetic
tone also are clearly needed. We can conclude, however,
that the increase in cardiac sympathetic tone in the late
stages of hamster cardiomyopathy appears to lead to a
817
shift in the rate-limiting step for cardiac norepinephrine
synthesis from the hydroxylation of tyrosine to the hydroxylation of dopamine. There is evidence that this shift,
which results in an accumulation of dopamine in the
noradrenergic nerve terminals of the heart, is a general
manifestation of augmented sympathetic nerve traffic
rather than a peculiarity of hamster cardiomyopathy.
Acknowledgments
Chlorisondamine was generously provided by the Ciba Pharmaceutical
Co., Summit, New Jersey.
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A possible change in the rate-limiting step for cardiac norepinephrine synthesis in the
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M J Sole, A B Kamble and M N Hussain
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Circ Res. 1977;41:814-817
doi: 10.1161/01.RES.41.6.814
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