1525
f3-Adrenergic Agonists Stimulate the
Oxidative Pentose Phosphate Pathway in the
Rat Heart
Heinz-Gerd Zimmer, Hans Ibel, and Ulrich Suchner
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The oxidative pentose phosphate pathway is poorly developed in the rat heart compared with
other organs, since the activity of glucose-6-phosphate dehydrogenase (G-6-PDH), the first and
rate-limiting enzyme of the oxidative pentose phosphate pathway, is low. As a consequence, the
available pool of 5-phosphoribosyl-l-pyrophosphate and the rate of adenine nucleotide
biosynthesis are limited. Isoproterenol, 24 hours after subcutaneous administration at 0.1, 1,
and 25 mg/kg, stimulated the activity of G-6-PDH in whole hearts dose-dependently from
4.3±0.16 (control) to 6.6± 0.35, 10.3+±0.82, and 11.5+±0.56 units/g protein, respectively. The
activity of 6-phosphogluconate dehydrogenase, another of the enzymes in the oxidative pentose
phosphate pathway, remained unchanged. G-6-PDH activity started to increase 12 hours after
isoproterenol application, when the glycogenolytic and functional response was over, and
reached a peak value between 24 and 48 hours. This stimulating effect was also demonstrated
in cardiac myocytes that were isolated 28 hours after isoproterenol application. /-Receptor
blockade with atenolol reduced the isoproterenol-induced increase in cardiac G-6-PDH activity
by 90%o. Cycloheximide, which inhibits translation, and actinomycin D, which interferes with
transcription, attenuated it by 83% and 78%, respectively. These results indicate that cardiac
/-adrenergic receptors and enzyme protein synthesis are involved in this effect. Other
/3-sympathomimetic agents such as dopamine, dobutamine, fenoterol, salbutamol, and terbutaline also stimulated myocardial G-6-PDH activity in a time- and dose-related manner. The
calcium antagonist D 600 (gallopamil) reduced the isoproterenol-elicited stimulation by 65%,
and verapamil blunted the fenoterol-induced increase by 50%6. This suggests that Ca2' ions also
contribute to the stimulation of the cardiac oxidative pentose phosphate pathway. (Circulation
Research 1990;67:1525-1534)
T he oxidative pentose phosphate pathway
(PPP) is important, since it is the link between carbohydrate and fatty acid as well as
purine and pyrimidine nucleotide metabolism (Figure 1). Glucose-6-phosphate (G-6-P), originating
from glycogenolysis or from glucose taken up by the
myocardial cell, is metabolized predominantly via
glycolysis. A certain portion, however, enters the
oxidative branch of the PPP of which glucose-6phosphate dehydrogenase (G- 6 -PDH) is the first and
rate-limiting enzyme.' This pathway has two major
functions to fulfill: 1) It provides reducing equivalents in the form of NADPH, which can be used for
the synthesis of free fatty acids and for the conversion
From the Department of Physiology, University of Munich,
F.R.G.
Supported by grants from the Deutsche Forschungsgemeinschaft (Zi 199/4-5, Zi 199/8-1).
Address for correspondence: Heinz-Gerd Zimmer, MD, Department of Physiology, University of Munich, Pettenkoferstr. 12,
D-8000 Munchen 2, F.R.G.
Received March 6, 1990; accepted August 2, 1990.
of oxidized glutathione to reduced glutathione. This
is important for the detoxification of reactive oxygen
species via glutathione peroxidase.2 2) In this pathway, ribose-5-phosphate is generated, which can be
transformed to 5-phosphoribosyl-1-pyrophosphate
(PRPP), and this is an essential precursor substance
for the synthesis of both pyrimidine and purine
nucleotides. There are connections between the oxidative PPP and glycolysis at two levels via the transaldolase and transketolase reactions.
It has been inferred that the capacity of the
oxidative PPP is low in the rat myocardium; this
inference is drawn from the fact that adenine nucleotide biosynthesis is small3 because of the limited
availability of PRPP.4 It has been shown for a variety
of species, including humans, that the activity of
myocardial G-6-PDH is low compared with that of
6-phosphogluconate dehydrogenase (6-PGDH).5
The critical step in the oxidative PPP, the G-6-PDH
reaction, can be bypassed with ribose, which elevates
via ribose-5-phosphate the PRPP pool and stimu-
1526
Circulation Research Vol 67, No 6, December 1990
GLYCOGEN
GLUCOSE-G -6 -P
2GSH
NADP*
GSSG
NADPH
F- 6 -P
,
F-1.6- p
6-PGL
l
FATTY
ACID
SYNTHESIS
LACTONASE
6-P-G
NADP+
8
)
\NAD~~NADPH
+H+
~~~~~6PGDH
<_ C02
I
Ru- 5 -P
__________GAP
RIBOSE _R-5
-p
PRPP
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PYRIMIDINE NUCLEOTIDE
SYNTHESIS
-
PYRUVATE
PURINE - NUCLEOTIDE
SYNTHESIS
FIGURE 1. Schematic representation of the oxidative pentose
phosphate pathway (center) and its connections to glycolysis
(right side) via the transaldolase and transketolase reactions
(arrows). G-1-P, glucose-i-phosphate; G-6-P, glucose-6phosphate; F-6-P, fructose-6-phosphate; F-1.6-P, fructose1,6-biphosphate; GAP, glyceraldehyde-3-phosphate; 6-PGL,
6-phosphogluconolactone; 6-P-G, 6-phosphogluconate; Ru5-P, ribulose-5-phosphate; R-5-P, ribose-5-phosphate; PRPP,
5-phosphoribosyl-1-pyrophosphate; GSH, reduced glutathione; GSSG, oxidized glutathione; G-6-PDH, glucose-6phosphate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase.
lates adenine nucleotide biosynthesis.6 It has been
demonstrated previously that an experimentally induced decline in the cardiac ATP pool can be
attenuated or even prevented by application of ribose.6-8
As to the regulation of this pathway, it is known
from studies on the liver that G-6-PDH is regulated
by the NADP+/NADPH ratio,1 which exerts a "fine
control." G-6-PDH is always inhibited by NADPH,9
and this inhibition can be overcome by oxidized
glutathione.' It has been demonstrated that the flow
through the oxidative PPP in the isolated perfused
rat heart can be enhanced acutely and rapidly by an
oxidizing agent that increases the oxidized glutathione level and the NADP+/NADPH ratio.10
It was the primary aim of this study to assess
quantatively the capacity of the oxidative PPP in the
rat heart compared with other organs. To do this, the
activity of G-6-PDH, the available pool of PRPP, and
the rate of adenine nucleotide biosynthesis were determined (Figure 1). The second aim was to examine
whether l3-adrenergic agonists"1 could influence the
oxidative PPP. These agents have already been shown
to activate glycogenolysis,12 to stimulate cardiac adenine nucleotide biosynthesis,4 and to have pronounced
positive chronotropic and inotropic effects.
Materials and Methods
The experiments were done on female SpragueDawley rats (200-250 g body wt) obtained from Mus
Rattus, Brunnthal, F.R.G. They were fed a control
rat chow diet (Altromin C 100, Altromin GmbH,
Lage, F.R.G.) with free access to tap water. The
substances were administered either as a subcutaneous injection or as a continuous intravenous infusion
via a catheter (Vygon, Aachen, F.R.G.) that was
positioned in the right jugular vein. The catheter was
connected to a 20-ml syringe placed in an infusion
pump (Infors AG, Basel, Switzerland). The infusion
rate was 4 ml/kg/hr.
Isoproterenol was purchased from Boehringer Ingelheim, Ingelheim, F.R.G., or Sigma Chemie
GmbH, Deisenhofen, F.R.G. It was dissolved in
0.9% NaCI containing ascorbic acid (0.2 g/l) and was
subcutaneously injected in different doses. Dopamine was kindly provided by Giulini Pharma, Hannover, F.R.G. It was administered as a subcutaneous
injection and as a continuous intravenous infusion.
Dobutamine was a gift from Lilly GmbH, Giessen,
F.R.G. Fenoterol was obtained from Boehringer
Ingelheim; terbutaline was kindly provided by Draco
Company, Lund, Sweden; and salbutamol was obtained from Kettelhack-Riker Pharma, Borken,
F.R.G. These substances were administered as continuous intravenous infusions containing ascorbic
acid. The syringes containing the substances were
protected against light. Compound D 600 (administered as a subcutaneous injection) and verapamil
(administered as a continuous intravenous infusion)
were donated by Knoll AG, Ludwigshafen, F.R.G.
[1-'4C]Glycine (specific activity, 54.2 mCi/mmol),
[8-14C]adenine (specific activity, 54.3 mCi/mmol),
and [U-14C]adenine (specific activity, 282 mCi/mmol)
were purchased from the Radiochemical Centre,
Amersham, U.K., and were injected into the tail vein.
Measurement of Metabolic Parameters
The activities of G-6-PDH (EC 1.1.1.49) and
6-PGDH (EC 1.1.1.44) were measured according to
the methods of Glock and McLean.13,14 After various
periods of in vivo exposure to the different substances, the rats were anesthetized with ether, and a
cannula was placed in the ascending aorta after
thoracotomy and tightly fixed there. The hearts were
quickly excised, and the coronary arteries were perfused via the cannula with an ice-cold KCI solution
(0.15 M containing 8 ml of 0.02 M KHCO3/l) to
remove blood and to stop the beating of the heart.
After homogenization of the hearts in the perfusion
medium, pH adjustment (7.0), and centrifugation
(ultracentrifuge model L5 -65, Beckman Instruments,
Inc., Fullerton, Calif.) at 20,000 rpm for 30 minutes,
the supernatants were dialyzed overnight. The enzyme activities were then measured spectrophotometrically. Protein concentration in the dialysate was
determined by using the modified biuret reaction.15
Zimmer et al Cardiac «-Receptors and Pentose Phosphate Pathway
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The mean specific activity of both enzymes was
expressed as units per gram protein.
Levels of cyclic AMP (cAMP) were determined
according to the method of Gilman.16 The content
of G-6-P was measured using the method of Lang
and Michal.17 The available pool of PRPP was
assessed from the radioactivity of adenine nucleotides after 5, 10, and 15 minutes of in vivo exposure
of the rats to intravenously injected [8-1'4C]adenine
or [U-14C]adenine. Since PRPP is consumed in the
reaction by which adenine is converted to AMP
catalyzed by adenine phosphoribosyltransferase
(EC 2.4.2.7) and since AMP is in rapid equilibrium
with ADP and ATP via the myokinase reaction (EC
2.7.4.3), the total radioactivity of the adenine nucleotides ATP+ADP+AMP is an indirect measure
of the available pool of PRPP.6 After the indicated
periods of vivo exposure, the hearts were excised in
ether anesthesia and frozen in liquid nitrogen.
After tissue extraction, adenine derived from adenine nucleotides by hydrolysis was separated, and
the radioactivity was counted in a tri-carb liquid
scintillation spectrometer (model 3380, Packard
Instruments, Downers Grove, Ill.).
The de novo synthesis (biosynthesis) of adenine
nucleotides was measured on the basis of the incorporation of [1-`4C]glycine (250 ,uCi/kg body wt injected into the tail vein) into adenine nucleotides.
After an in vivo exposure time of 60 minutes, the
hearts were excised in ether anesthesia and frozen in
liquid nitrogen; the radioactivity was measured after
extraction, separation, and purification of the adenine nucleotides, according to methods described
earlier.3 Rates of de novo synthesis were calculated
by relating the total radioactivity of adenine nucleotides due to the incorporation of [1-14C]glycine to the
mean specific activity of the tissue glycine precursor
pool, which was determined with an amino acid
analyzer (model BC 200, BioCal Munchen, F.R.G.).
Rates of protein synthesis were determined in the
same hearts in which adenine nucleotide biosynthesis
was measured by relating the radioactivity of total
cardiac proteins to the mean specific activity of the
amino acid precursor pool.'8
Measurement of Left Heart Function
When the function of the left heart was measured,
the rats were anesthetized with 80 mg/kg i.p. thiobutabarbital sodium (Inactin, Byk Gulden, Konstanz,
F.R.G.). The depth of anesthesia was tested by eliciting reflexes in the legs by forceps. After tracheotomy,
a catheter was placed in the trachea to maintain
airway patency, to allow suction of secretions, if necessary, and to institute artificial respiration in case of
an emergency.
The ultraminiature catheter pressure transducer
(model PR-249, Millar Instruments, Inc., Houston)
was used for left heart catheterization.19 This catheter was inserted into the right carotid artery and
advanced upstream from the aorta into the left
ventricle. It was attached to a Millar control unit
1527
(model TC-100), which was connected to an HSE
electromanometer (Hugo Sachs Elektronik, MarchHugstetten, F.R.G.) for the measurement of left
ventricular (LV) systolic pressure. The maximal rate
of rise in LV pressure (LV dP/dtma,) was obtained
with an electronic differentiation system (PhysioDifferentiator, Hugo Sachs Elektronik). The recording system was a Brush 2600 recorder (Gould Inc.,
Cleveland). Calibration of pressure and LV dP/dtm,
was done using a mercury manometer (type 376,
Hugo Sachs Elektronik) and the calibration device
that is built into the Physio-Differentiator. When the
catheter was positioned in the left ventricle, there
was no pressure gradient across the aortic valve: LV
diastolic pressure did not increase, even when the
catheter was in place for hours; cardiac output measured with the thermodilution technique was essentially unchanged when the catheter was positioned in
the left ventricle; and systolic aortic pressure was as
high as LVSP when the catheter tip was withdrawn
from the left ventricle and placed in the aorta.
Isolation of Cardiac Myocytes
Rats were anesthetized with thiopental sodium
(Trapanal, Byk Gulden) and heparinized 28 hours
after subcutaneous injection of NaCI or isoproterenol
(25 mg/kg). The chest was opened, and the hearts
were quickly removed. The aorta was immediately
cannulated and attached to a modified Langendorff
apparatus. Isolated myocytes were prepared by perfusion with collagenase type II obtained from Sigma
Chemie GmbH in calcium-free Joklik medium as
described by Bishop and Drummond.20 The right
ventricle was separated from the septum by sharp
dissection and minced into calcium-free Joklik media
containing 0.01 mM EGTA, poured through nylon
mesh (250 ,tm), and fixed in glutaraldehyde for visual
quality control of the isolation procedure using an
Axioskop microscope (Zeiss, Oberkochen, F.R.G.).
The left ventricles of hearts from two identically
treated rats were minced immediately into the KCI
solution (0.15 M containing 8 ml of 0.02 M KHCOJ1)
that was used for homogenization and poured
through nylon. After correcting the pH and centrifugation, the supernatants were dialyzed overnight.
G-6-PDH activity was then measured spectrophotometrically, as described earlier ("Measurement of
Metabolic Parameters").
Statistical Analysis
All values are expressed as mean±SEM. For calculating significance, Student's t test for unpaired
data was used. Differences were considered significant at a value of p<0.05.21
Results
It was the purpose of the first series of experiments
to estimate the capacity of the oxidative PPP in
several rat organs. The activity of G- 6-PDH, the first
and regulating enzyme, the available pool of PRPP,
and the rate of purine nucleotide biosynthesis were
1528
Circulation Research Vol 67, No 6, December 1990
TABLE 1. Effect of Isoproterenol on the Activities of Glucose-6phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase in Rat Hearts 24 Hours After Subcutaneous Injection of
Three Different Doses
n
G-6-PDH
(units/g protein)
4.3±0.16
6-PGDH
(units/g protein)
11.2+0.25
28
Control
ISO
11.3±0.33
7
6.6±0.35*
0.1 mg/kg
12.3+0.52
11
10.3+0.82*
1.0 mg/kg
12.2±0.46
12
11.5+0.56*
25.0 mg/kg
Values are mean± SEM; n =number of experiments; G-6-PDH,
glucose-6-phosphate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase; ISO, isoproterenol.
*p<0.0005 vs. control.
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FIGURE 2. Bar graphs showing activities ofglucose-6-phosphate dehydrogenase (top panel), assessment of the available
pool of 5-phosphoribosyl-1-pyrophosphate (PRPP) measured
as incorporation of ['4C]adenine into adenine nucleotides
after an in vivo exposure time of 15 minutes (middle panel),
and rates of adenine nucleotide biosynthesis (bottom panel)
in various organs of normal rats (open bars) and the effect of
ribose on the available PRPP pool and on adenine nucleotide
biosynthesis (hatched columns). Data are mean ±SEM; number of experiments is indicated in parentheses. In experiments
in which ribose was administered, it was infused continuously
for 4 hours (200 mg/kg/hr), when the PRPP availability was
measured; adenine nucleotide biosynthesis was measured 60
minutes after an intravenous injection of ribose (100 mg/kg).
measured (Figure 2). There was a parallel behavior
of these three parameters in the organs examined.
They were all highest in kidney, followed by liver,
heart, and skeletal muscle. From these results, it
appears that the capacity of the oxidative PPP is
lowest in heart and skeletal muscle. To substantiate
this, ribose was administered. It did not alter the
PRPP availability and adenine nucleotide biosynthesis in kidney and liver. In heart and skeletal muscle,
however, there was a marked increase (Figure 2,
middle and bottom panels).
It was now of interest to determine whether the
oxidative PPP in the heart could be enhanced in
intact rats so that more PRPP would be provided for
nucleotide metabolism. In a first attempt, the effect
of isoproterenol on the activity of cardiac G-6-PDH
and 6-PGDH was tested. Isoproterenol stimulated
the activity of G-6-PDH in a dose-dependent man-
ner but had no influence at all on the activity of
6-PGDH (Table 1).
The changes in metabolic and functional parameters that occurred over a time period of 72 hours
subsequent to the highest dose of isoproterenol are
shown in Figure 3. There was an immediate increase
in the cAMP content as well as in the content of
G-6-P. Parallel to these metabolic alterations, heart
rate and LV dP/dtmx were also elevated. After 12 and
24 hours, these metabolic and functional parameters
had returned to the control level. However, adenine
nucleotide biosynthesis, which was also enhanced
very early, remained elevated, although at a somewhat lower level. With a time lag there was an
increase in cardiac protein synthesis, and when this
was clearly enhanced, the activity of myocardial
G-6-PDH started to be stimulated. The peak was
reached quite late, after 24 and 48 hours.
After 28 hours of in vivo exposure to isoproterenol
(25 mg/kg, subcutaneously applied), G-6-PDH activity
was measured in freshly isolated cardiac myocytes
obtained from the left ventricle and compared with that
of NaCl-injected rats. In myocytes from control rats, it
was 0.3+±0.05 units/g protein (n=7); in those from
isoproterenol-treated rats, it was significantly increased
(p<0.0005) to 3.2+0.55 units/g protein (n=6).
From the time course (Figure 3), it is suggestive
that activation of G- 6-PDH in the heart is dependent
on protein synthesis and thus may reflect an increased new synthesis of enzyme protein. Therefore,
the effect of inhibitors of protein synthesis on G-6PDH was examined. Cycloheximide and actinomycin
D, which had no effect on cardiac G-6-PDH activity
by themselves, attenuated the isoproterenol-induced
increase by 83% and 78%, respectively (Table 2).
/3-Receptor blockade with atenolol reduced the
isoproterenol-elicited enhancement of G-6-PDH activity by 90%. The calcium antagonist compound D
600 (gallopamil) blunted it by 65% (Figure 4). When
dopamine was administered either as subcutaneous
injection or as continuous intravenous infusion, there
was a time- and dose-dependent stimulation of cardiac G-6-PDH activity, which was almost completely
abolished with atenolol (Table 3). Dobutamine also
Zimmer et al Cardiac a-Receptors and Pentose Phosphate Pathway
1529
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TABLE 2. Activity of Myocardial Glucose-6-phosphate Dehydrogenase After a Single Subcutaneous Injection of Isoproterenol and
the Effect of Cycloheximide and Actinomycin D in Control and
Isoproterenol-Treated Rats
G-6-PDH
n
(units/g protein)
Control
28
4.3+0.16
Iso
12
11.5+0.56*
Cycloheximide
3
4.4±0.17
Actinomycin D
4
4.9±0.29
5
ISO+cycloheximide
5.5±0.21t
6
ISO+actinomycin D
5.9±0.40t
Values are mean±SEM; n=number of experiments. G-6-PDH,
glucose-6-phosphate dehydrogenase; ISO, isoproterenol. ISO was
administered in a dose of 25 mg/kg. Cycloheximide (0.6 mg/kg)
and actinomycin D (0.3 mg/kg) were injected intraperitoneally for
the first time 6 hours before ISO administration. The same doses
of cycloheximide and actinomycin D were given 6 and 12 hours
after ISO injection. All measurements were made 24 hours after
ISO administration.
*p<0.0005 and tp<0.025 vs. control.
nucleotides was determined under the influence of
isoproterenol, dopamine, and dobutamine. Isoproterenol increased the incorporation of labeled adenine both after 5 and 24 hours, subsequent to subcutaneous injection. Also, dopamine and dobutamine
stimulated this process after 48 hours of continuous
intravenous infusion (Figure 5).
Since dopamine and dobutamine are used clinically, it was of interest whether other sympathomimetic agents that are given to patients may affect the
cardiac PPP. Fenoterol, salbutamol, and terbutaline
stimulated G-6-PDH activity, though to different
CONTROL
ISO (lmg/kg)
|*ATENOLOL
D600
*D60
(12)
12'
10
1 5 12
24
48
ISOPROTERENOL (25mg/kg,s.c.)
1SO( 25 mg /kg)
*
72 h
FIGURE 3. Graphs showing effects of isoproterenol on the
levels of cardiac cyclic AMP (cAMP, *-*) and glucose-6phosphate (G-6-P, *--s) (top panel), on heart rate (*-*)
and left ventricular (LV) dP/dt. (v---4) (second panel from
top), on myocardial adenine nucleotide synthesis (middle
panel), on protein synthesis (second panel from bottom),
and on the activity of cardiac glucose-6-phosphate dehydrogenase (G-6-PDH) (bottom panel) in rats. Values are
mean ±SEM; the number of animals at each point in time is
given in parentheses.
stimulated G-6-PDH activity, though not as markedly as did dopamine (Table 3).
To examine whether the available pool of PRPP
may also be affected by these substances, the incorporation of [14C]adenine into myocardial adenine
c
&;
8
26
(8
-~
(28)
+Vi
LI
4
2J
I
* p<0.0005 vs. CONTROL
-tp'0.025 vs. CONTROL
FIGURE 4. Bar graph showing activity of myocardial glucose-6-phosphate dehydrogenase 24 hours after subcutaneous
injection of isoproterenol (ISO) alone and in combination
with the (-receptor blocker atenolol or the calcium antagonist
compound D 600 (gallopamil). Atenolol (1 mg/kg s.c.) and D
600 (10 and 5 mg/kg s.c.) were given twice, 12 hours apart.
Values are mean ±SEM; number of experiments is indicated in
parentheses.
Circulation Research Vol 67, No 6, December 1990
1530
TABLE 3. Influence of Dopamine and Dobutamine on the Activity of Cardiac Glucose- 6-phosphate
Dehydrogenase in Rats
G-6-PDH
Time period
n
(hr)
(units/g protein)
4.5 +0.17
14
Control
5.8+0.33*
24
4
Dopamine (500 mg/kg s.c.)
8
48
Dopamine (500 mg/kg s.c.)
10.3+0.96t
5.0+0.45
4
48
Dopamine (500 mg/kg s.c.)+atenolol
5.7+0.25*
48
5
Dopamine (1 mg/kg/hr i.v.)
48
9
Dopamine (10 mg/kg/hr i.v.)
8.3±0.54t
11
48
6.0±0.23*
Dobutamine (10 mg/kg/hr i.v.)
Values are mean+SEM; n=number of experiments. G-6-PDH, glucose-6-phosphate dehydrogenase. Dopamine was
administered as a- single subcutaneous injection or as continuous intravenous infusion. Dobutamine was applied only
as continuous intravenous infusion. Atenolol was injected subcutaneously in a dose of 10 mg/kg three times.
Measurements of G-6-PDH activity were made after the indicated periods of time.
*p<0.025 and tp<0.0005 vs. control.
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degrees (Figure 6). None of these substances had an
influence on cardiac 6-PGDH activity (data not
shown).
Since fenoterol had the most marked effects, further experiments were done with this drug. Fenoterol
stimulated cardiac G-6-PDH activity in a dose-dependent manner (Figure 7). To examine whether this
increase can be influenced by blocking the calcium
channel, the calcium antagonist verapamil was used.
Verapamil antagonized the hemodynamic response
to fenoterol effectively. Heart rate and LV systolic
pressure were even lower than control values when
verapamil was administered in combination with
fenoterol (Figure 8). Yet, G-6-PDH activity was still
increased by 30% in the presence of verapamil, while
the pressure-rate product was even lower than control values (Figure 9).
X103
z
:X
200-
LL
Discussion
Our comparative studies have shown that the capacity of the oxidative PPP was lower in heart and skeletal
muscle than in kidney and liver. The assessment of this
pathway was based on three independent parameters,
G-6-PDH activity, PRPP availability, and adenine
nucleotide biosynthesis, which were measured in different rats. The magnitude of these parameters corresponded very closely in each rat organ examined (Figure 2). The G-6-PDH activity was lower in the
myocardium than in the kidney and liver.14 This supports earlier findings obtained in dog heart muscle
homogenates,22 in isolated cardiac myocytes from
rats,23 and in the rat during development.24 In the latter
study, the activities of both G-6-PDH and 6-PGDH
were always lower in the heart than in the liver.
The first and rate-limiting enzyme, G-6-PDH, seems
to be responsible for the limited capacity of the oxida-
Iso
24 h
0
,0°
15000
0
0
9'
100
50-
4c
5
10
15
o/
A-/
0'
/
A
5
10
15
5
10
15
5
10
15
min
FIGURE 5. Graphs showing radioactivity of cardiac adenine nucleotides (AN) due to the incorporation of [14Cladenine 5 and 24
hours after subcutaneous injection of 25 mg/kg isoproterenol (ISO) and after 48 hours of continuous intravenous infusion of
dopamine and dobutamine (10 mg/kglhr each) in rats. Open circles indicate infusion of agent; closed circles indicate control
condition. [8-'4C]Adenine (0.25 mCi/kg) was used 5 hours after ISO application; [U-14Cladenine (0.1 mCi/kg), 24 hours after
ISO; [U-14C]adenine (0.075 mCi/kg), 48 hours after dopamine; and [U-14Cladenine (0.04 mCilkg), 48 hours after dobutamine.
Measurements were made in individual animals after 5, 10, and 15 minutes, respectively, subsequent to intravenous injection of
[14C]adenine.
Zimmer et al Cardiac fl-Receptors and Pentose Phosphate Pathway
CONTROL
FENOTEROL SALBUTAMOL TERBUTALINE
(8)
8-
(6)
( 5)
6.C:
(50)
0
0)
4.
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
$p <
0.0005
FIGURE 6. Bargraph showing effect offenoterol, salbutamol,
and terbutaline (1 mg/kg/hr each), administered as a continuous intravenous infusion for 48 hours, on the activity of cardiac
glucose- 6-phosphate dehydrogenase in rats. Values are
mean ±SEM; number of animals is indicated in parentheses.
tive PPP in the heart, so that the available pool of
PRPP and the rate of adenine nucleotide biosynthesis
are very small.4 This limitation can be overcome by
ribose, which bypasses the critical step, the G-6-PDH
reaction. As a consequence, the PRPP pool and the
rate of adenine nucleotide biosynthesis were elevated
by ribose (Figure 2).6,25 Ribose has been shown to
restore, partially or entirely, the adenine nucleotide
pool in various pathophysiological situations, such as
stimulation with isoproterenol,6 postischemic recovery,7
and myocardial infarction.8
CONTROL
--1Qlmgkg/h
FENOTERO
1 mg/kg/h
10 mg/kg/h
(6)
10-
(8)
8-
(5)
c
0
L-
6-
(50)
4-
2-
FIGURE 7. Bar graph showing dose-related increase in the
activity of cardiac glucose-6-phosphate dehydrogenase in rats
after 48 hours of continuous intravenous infusion offenoterol.
Values are mean ±SEM; number of animals is indicated in
parentheses.
1531
In skeletal muscle, G- 6-PDH activity was even
lower than in the heart,26 and adenine nucleotide
biosynthesis was not measurable under control in
vivo conditions. When ribose was administered, it
was of the same order of magnitude as in the heart in
the control situation (Figure 2). The conditions for
measuring adenine nucleotide biosynthesis may be
different in the isolated rat extensor digitorum longus
muscle27 and in the perfused rat hindquarter preparation.28 In these preparations, adenine nucleotide
biosynthesis was readily measurable.
That G-6-PDH is the rate-limiting enzyme of the
oxidative PPP in the heart is also supported by our
results obtained with isoproterenol. This ,B-adrenergic agonist induced a dose-dependent increase in
G- 6-PDH activity but did not affect 6-PGDH activity
(Table 1). Parallel to the increase in G-6-PDH
activity, the PRPP pool was elevated (Figure 5), and
adenine nucleotide biosynthesis was enhanced (Figure 3). Our time course studies, however, revealed
that the temporal relations were more complex.
From the data in Figure 3, it appears that there were
two distinct phases subsequent to fl-receptor stimulation: The first phase, lasting about 5 hours, was
characterized by the enhancement of glycogenolysis
as evidenced by the elevation of cardiac cAMP and
G-6-P levels, by the positive chronotropic and inotropic response, by the elevation of the PRPP pool
(Figure 5), and by the immediate increase in adenine
nucleotide biosynthesis. There was no change in
G-6-PDH activity (Figure 3). During the second
phase, after 12 hours subsequent to isoproterenol,
glycogenolysis and heart function had become normalized, but adenine nucleotide biosynthesis was still
increased, though at a lower level. The PRPP pool
was also elevated (Figure 5), and protein synthesis as
well as G-6-PDH activity started to increase steeply
(Figure 3). Thus, the increase in the available PRPP
pool and in adenine nucleotide biosynthesis coincided with the stimulation of glycogenolysis during
the first phase and with the enhancement of G-6PDH activity during the second phase.
Since G-6-PDH activity was unchanged during the
first phase, a mechanism other than an enhanced
flow through the oxidative PPP may be responsible
for the increase in the PRPP pool and in adenine
nucleotide biosynthesis. One possibility is that not
only glycogenolysis but also glycolysis is stimulated by
isoproterenol. This could then lead to an increased
availability of PRPP through the nonoxidative PPP
via the transaldolase and transketolase reactions.
The activities of these enzymes were shown to be
much higher than those of G-6-PDH and 6-PGDH
in muscle.26 Furthermore, the large amounts of
NAD+ present in the heart (29) may favor glycolysis,
whereas the small quantity of NADP+ may limit the
oxidative PPP. In this context it is interesting to
mention that triiodothyronine did not affect cardiac
G-6-PDH activity,30 yet the cAMP level as well as
adenine nucleotide and protein synthesis
were in-
Circulation Research Vol 67, No 6, December 1990
1532
U
(j)
0)
0
-0
0,
I:
E 300
E
E
E
E
200-
±tp<0.005 vs.CONTROL
*p<0.0005;
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
creased,31 suggesting stimulation of glycolysis and of
the nonoxidative branch of the PPP.
Our time-course study (Figure 3) also provided some
hints as to the mechanism for the increase in G-6-PDH
activity. G-6-PDH activity did not increase before
protein synthesis was enhanced. From this temporal
relation it can be suggested that activation of G-6-PDH
in the heart depends on protein synthesis and thus may
reflect an increased new synthesis of enzyme protein.
Both interference of transcription with actinomycin D32
and inhibition of translation by cyclohexinmide attenuated the isoproterenol-induced G-6-PDH stimulation
by about 80% (Table 2). Thus, it appears that synthesis
of new enzyme protein, that is, enzyme induction, is
involved in the observed stimulation of G-6-PDH activity. But what is the mechanism for the enhancement
of protein synthesis? In this regard, the f3-receptormediated cAMP elevation seems to play an important
role. When the increase in cAMP was prevented by
FENOTEROL
CONTROL
_
FENOTEROL
+
VERAPAMIL
~~~~~~(36)
o
*t
C
(2)
U.CD.
0
-
~~~~~
80000
~~~~~(14.)
(9)
(8)
6-
60000 I
E
E
~~~~~~~~~~~~~~~(8)
-40000
O~'
-20000
>
IIV
u)
[]
FIGURE 8. Bar graph showing
left ventricular (LV) functional
parameters after 48 hours of continuous intravenous infusion of
fenoterol (1 mg/kg/hr) alone
(hatched columns) and in combination with verapamil (4 mg/kg!
hr) (double-hatched columns)
compared with controls (open
columns). LVSP. LV systolic
pressure. Values are mean +SEM;
number of rats is indicated in
parentheses.
_005
tp <0.005 vs.Control
FIGURE 9. Bar graph showing effects of fenoterol (1 mg/kg!
hr) alone and in combination with verapamil (4 mg/kg/hr),
administered as continuous intravenous infusion in rats for 48
hours, on the activity of myocardial glucose-6-phosphate
dehydrogenase (G-6-PDH) (open columns) and on the left
ventricular systolic pressure-heart rate product (LVSPxHR)
(hatched columns). Values are mean +SEM; number of animals is indicated in parentheses.
fl-blockers, the isoproterenol-induced enhancement in
adenine nucleotide and protein synthesis3' and in G-6PDH (Figure 4) was almost entirely prevented. Also,
the dopamine-elicited stimulation was abolished by
atenolol (Table 3). Therefore, it may be that ,B-adrenergic agonists, via elevation of intracellular levels of
cAMP, induce the transcription of the respective genes
by activating a cAMP-dependent protein kinase and by
phosphorylation of a protein factor that links kinase
activation with transcription of cAMP-responsive
genes.33,34 It is interesting that the catecholamines
epinephrine and isoproterenol also stimulated the oxidative PPP in the monkey palm eccrine sweat gland and
that this could be suppressed by the ,8-receptor blocker
propranolol.35
Apart from activation of the adenylate cyclasecAMP system, Ca'+ ions seem also to contribute to
the stimulation of G-6-PDH activity. When the calcium antagonists D 600 (gallopamil) and verapamil
were applied in combination with isoproterenol or
fenoterol, the catecholamine-induced increase in
G-6-PDH activity was attenuated by 65% (Figure 4)
and 50% (Figure 9), respectively. Similarly, D 600
blunted both the isoproterenol-induced increase in
cAMP36 and in adenine nucleotide biosynthesis by
50%.4 The remaining stimulation of G- 6-PDH activity occurred independent of the function of the heart:
The isoproterenol-mediated stimulation of G- 6PDH activity took place at a time when the positive
chronotropic and inotropic effect was over (Figure
3). Moreover, the fenoterol-induced increase in heart
function was abolished by verapamil to such an
extent that heart rate and LV systolic pressure (Figure 8) and the pressure-rate product (Figure 9) were
even lower than control, yet the G-6-PDH activity
was still elevated. Thus, there was a dissociation
between function and metabolism of the heart in
these experimental conditions.
It is interesting that not only isoproterenol but also
clinically applied sympathomimetic substances stimulate the oxidative PPP. Dopamine37 and dobutamine38
were applied for 24 or 48 hours, since the time-course
studies with isoproterenol had revealed the peak G-6PDH activity to be during this period of time (Figure 3).
Zimmer et al Cardiac a-Receptors and Pentose Phosphate Pathway
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
These agents act directly at cardiac fl1-receptors. Accordingly, the effect of dopamine on myocardial G-6PDH activity was prevented by the f1-selective blocker
atenolol (Table 3). Also, sympathomimetic agents that
interact with /32-receptors,39 such as fenoterol,40 salbutamol,41 and terbutaline, and are used for the treatment
of bronchoconstrictive disease42-stimulated G-6-PDH
activity after an in vivo exposure time of 48 hours
(Figures 6 and 7). Considering the high doses that were
applied in our studies, this effect may be due to the
influence on cardiac /31-receptors. Apart from their
positive inotropic effect, all these sympathomimetic
agents had metabolic effects that ultimately tend to
restore the cardiac ATP pool (Figure 1). This can be
considered as a beneficial effect in the treatment of
heart failure.43
A final aspect concerns the cellular compartment in
which the oxidative PPP is localized within the myocardium. It has been shown in this study that G-6-PDH
activity was present in freshly isolated cardiac myocytes
and that it was increased about 10-fold after 28 hours
of in vivo exposure to a high dose of isoproterenol.
Since G-6-PDH activity was lower in isolated cardiac
myocytes compared with whole heart, other cell elements in the myocardium must also have this enzyme.
The coronary endothelial cells can be excluded as a
potential site, since the biosynthesis of adenine nucleotides, which is dependent on the oxidative PPP, was
inhibited by isoproterenol.44 A likely cellular compartment may therefore be the connective tissue. Since
isoproterenol in the doses applied induces focal myocardial cell lesions,45 the observed increase in G-6PDH could well have occurred in inflammatory or
connective tissue cells. It is known that the oxidative
PPP is increased markedly subsequent to experimental
myocardial infarction.46 However, when the isoproterenol-induced focal myocardial injury was prevented by
simultaneous administration of a calcium antagonist,47
G-6-PDH activity was still enhanced (Figure 4). Furthermore, the isoproterenol-induced percent increase
was much higher in isolated cardiac myocytes than in
the whole heart. That the oxidative PPP is active in
isolated cardiac myocytes and that it can be altered
experimentally has also been shown in a previous
study.23
In summary, all 3-adrenergic agonists tested stimulated the oxidative PPP in the rat heart in vivo in a
dose- and time-related manner. An increase in the
oxidative PPP has also been observed during the
development of cardiac hypertrophy due to aortic
constriction.3048,49 Interestingly, other positive inotropic agents such as ouabain (authors' unpublished
observations) and triiodothyronine had no effect.30
Therefore, the stimulation of the oxidative PPP
seems to be a new metabolic effect that is characteristic for sympathomimetic agents.
Acknowledgments
The excellent technical assistance of Ms. G. Steinkopff and Mrs. B. Sender is gratefully acknowledged.
1533
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KEY WoRDs * adenine nucleotide biosynthesis * catecholamines
heart function * glucose-6-phosphate dehydrogenase
ribose
a
Beta-adrenergic agonists stimulate the oxidative pentose phosphate pathway in the rat
heart.
H G Zimmer, H Ibel and U Suchner
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Circ Res. 1990;67:1525-1534
doi: 10.1161/01.RES.67.6.1525
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