Download The Effect of Intracellular Oxygen Concentration on Lactate Release

448
The Effect of Intracellular Oxygen Concentration on
Lactate Release, Pyridine Nucleotide Reduction, and
Respiration Rate in the Rat Cardiac Tissue
Ryuichiro Araki, Mamoru Tamura, and Isao Yamazaki
From the Biophysics Division, Research Institute of Applied Electricity, Sapporo Japan,
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SUMMARY. By measuring the absorbance change due to myoglobin oxygenation in hemoglobinfree isolated perfused rat hearts, we analyzed effects of perfusion pressure and heart rate upon
the intracellular oxygen concentration. With Langendorff perfusion, the cardiac tissue was kept
normoxic (above 50 ^M O2) at aortic pressure above 50 cm H2O, but became hypoxic (8 /XM O2) at
30 cm H2O. The increase in cardiac work, expressed as the product of peak systolic pressure and
heart rate, increased oxygen consumption at aortic pressure of 50-200 cm H2O. The heart was
kept normoxic under these conditions. Lactate release, oxygen consumption, and the oxidationreduction state of pyridine nucleotide were measured as a function of myoglobin oxygenation
under various normoxic and anoxic conditions. Pyridine nucleotide fluorescence and lactate
release started to increase as the intracellular oxygen concentration decreased to 6 and 10 ^M,
respectively. Oxygen consumption was kept constant until the oxygen concentration decreased to
10 HM and slowed down below it. A close relationship between oxygen consumption and lactate
release was observed. Infusions of epinephnne and norepinephrine under normoxic perfusion
conditions increased cardiac work, oxygen consumption, and lactate release. More than 50% of
myoglobin was then deoxygenated even under normoxic perfusion conditions. The increase in
lactate release was ascribable to the increase in glycolytic flux caused by hypoxia. The change of
pyridine nucleotide fluorescence by epinephrine was also explained by hypoxia in cardiac tissue.
(Circ Res 53: 448-455, 1983)
THE function of mammalian cardiac tissue is exquisitely sensitive to oxygen. Under hypoxic conditions, its mechanical performance declines rapidly,
and the integrity of the tissue is compromised. This
is due to the fact that myocardial energy production
depends predominantly on the aerobic metabolism
through /3-oxidation of fatty acid (Neely and Morgan, 1974). ATP synthesis through glycolysis in an
anoxic state is insufficient to maintain cardiac performance at its normoxic level, although the glycolytic flux is significantly increased (Williamson,
1966a; Opie, 1976). Therefore, the intracellular oxygen concentration is crucial for normal function.
The oxygen concentration in various tissue has
been measured polarographically with a microelectrode (Broelsh et al., 1978; Silver, 1979). Optical
methods have also been developed by several investigators (Longmuir and Knoop, 1976; Millikan,
1937; Chance, 1976; Jobsis, 1977; Opitz et al., 1978).
For instance, emission of fluorescence at 470 run
from excitation light at 340 nm can be used to detect
changes in the content of reduced pyridine nucleotides. Chance and Schoener (1962) have studied
correlations between the electrical activity of the
cortex and the degree of pyridine nucleotide reduction caused by the transition from aerobiosis to
anoxia. The dependence of the oxidation-reduction
(redox) state of pyridine nucleotide on the oxygen
concentration is calibrated with pigeon heart mitochondria by Sugano et al. (1974), who has reported
that half-maximal reduction of pyridine nucleotide
occurs at an oxygen concentration of 0.08 MM in state
3. Similarly, half-maximal reduction of cytochrome
a + a3 occurs at oxygen concentrations of 0.3 /*M in
state 3 and less than 0.07 ^M in state 4 (Oshino et
al., 1974). These observations suggest the existence
of a critical oxygen concentration, at which point
respiration kinetics change from zero-order to firstorder with respect to the oxygen concentration
(Longmuir, 1957; Jobsis, 1972). The concentration is
below 1 mm Hg in mitochondria and above 10 mm
Hg in several tissues (Chance, 1965; Wittenberg,
1970). The difference may be explained, in part, by
the gradient of oxygen concentration between cytosolic and mitochondrial compartments (Tamura et
al., 1978).
Because of simplicity and convenience, the hemoglobin-free isolated heart perfused by the Langendorff technique has been used widely in the studies
of myocardial energy metabolism. The fundamental
characteristics of the preparation has been examined
under various conditions (Williamson, 1964; Neely
et al., 1967). The effects of various hormones and
drugs on the energy metabolism have also been
studied with this preparation, and the phosphorylation of some enzymes and membrane constituents
Araki et a/./Myocardial Tissue Oxygenation
is observed when catecholamine is added (Williamson, 1966b; Hiraoka et al., 1980).
Since myoglobin is located in cytosolic space of
the heart, its oxygenated degree reflects directly the
oxygen concentration in the tissue (Tamura et al.,
1978, 1983). We have measured the oxygenation
state of myoglobin in the perfused rat heart by
reflectance spectroscopy and determined the quantitative relation of the intracellular oxygen concentration with respiration rate, lactate release, and
fluorescence of pyridine nucleotide under various
experimental conditions. The detailed results are
reported in this paper.
Methods
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Hemoglobin-Free Isolated Perfused Heart
Male albino rats of the Wistar strain (230-260 g body
weight), fed on a commercial diet, were used. Rats were
neither heparinized nor anesthetized throughout the experiments. Perfusion was carried out by a slightly modified
method of Langendorff. The perfusion apparatus was
essentially the same as that reported previously (Tamura
et al., 1978). The aortic pressure was kept constant at 80
cm H2O unless otherwise noted. The perfusate contained
10 mM glucose and 2.5 ITIM calcium, equilibrated with a
gas mixure of 95% O2 + 5% CO2 for normoxic, or 95%
N2 + 5% CO2 for anoxic, conditions. The perfusate was
not recycled in a flow-through system, and its temperature
was maintained at 27°C. After equilibration for 15 minutes, hearts were subjected to the anoxic condition for
about 1 minute by changing the oxygen-saturated perfusate to the nitrogen-saturated one. Changes in absorbance
of myoglobin and fluorescence intensity of pyridine nucleotide for this normoxic-anoxic transition were full scale.
Spectrophotometric Measurement of the Perfused
Heart
Dual-wavelength reflectance spectrophotometry was
used to measure myoglobin oxygenation (Tamura et al.,
1978, 1983). Light longer than 560 nm was guided onto
the surface of left ventricle of perfused heart with a flexible
light guide 1 cm in diameter. The light reflected from the
heart was again guided into photomultipliers with a Yshaped light guide through a pair of interference filters.
Measurement and reference wavelengths were 580 and
620 nm, and the difference in absorbance between the
above two wavelengths was recorded. The oxidationreduction state of pyridine nucleotide was measured by
slightly modified surface fluorometry, as described by
Chance et al. (1965). The excitation light at 360 nm was
guided into the left ventricle of perfused heart with a
quartz light guide to excite reduced pyridine nucleotide in
the tissue. The emitted light was guided into a photomulriplier for fluorescence measurement through a filter centered at 470 ± 20 nm. A part of the reflected light was
guided into another photomultiplier, the output from
which was used to subtract the artifact from the fluorescence signal with a difference amplifier.
Heart Contractility and Cardiac Work
The left ventricular pressure (LVP) was measured by
using a microtip pressure transducer (model PC-350, Millar Instrument). A 22-gauge needle was inserted into the
449
left ventricle through the wall, and the pressure change
was transmitted through a 1 -mm diameter vinyl tube filled
with perfusate to a pressure transducer. The first derivative of left ventricular pressure change (dP/dt) was obtained electrically using a differentiating circuit with a
time constant of 20 msec.
Heart rate was measured with a frequency counter fed
on the output of LVP or R wave of electrocardiogram
(ECG).
Cardiac work was expressed as the product of peak
systolic pressure and heart rate (mm Hg/min) according
to the method of Kobayashi and Neely (1979).
Coronary Flow
The flow rate was measured continuously with an
electromagnetic flow meter (MFV-110, Nihon Koden) with
a flow probe placed in the perfusion circuit.
Oxygen Concentration in Effluent Perfusate, and
Oxygen Consumption
A Tygon tube with a diameter of 1 mm was inserted
into the pulmonary artery, and a portion of the effluent
perfusate was pumped through the electrode chamber at
a constant rate of 1 ml/min. A Clark-type oxygen electrode was inserted into the chamber. The inflowing oxygen
concentration was measured similarly with a 22-gauge
needle inserted into the perfusion circuit. The rate of
oxygen consumption was calculated as a product of flow
rate and difference in the oxygen concentration between
inflowing and effluent perfusates.
Assay of Lactate in the Effluent Perfusate
For determination of lactate release from the heart, the
effluent perfusate from the pulmonary artery was collected in the test tube for 4 seconds at appropriate time
intervals. The concentration of lactate was determined
enzymatically (Gutman and Wahlefeld, 1974) and the
inflowing perfusate was used as reference.
Materials
The enzymes for the lactate assay were purchased from
Boehringer Mannheim Gmbh. Epinephrine hydrochloride
and dopamine hydrochloride were obtained from Sigma
and Nakarai Chemicals, respectively. All other chemicals
were of analytical reagent grade.
Results
Effects of Perfusion Pressure and Heart Rate on
the Heart Function
Figure 1 shows the effect of perfusion pressure on
absorbance of myoglobin and various parameters
characterizing the cardiac performance. The absorbance change due to an aerobic-anaerobic transition
was shown at the right of the figure for calibration.
The change corresponded to the full scale for deoxygenation of myoglobin because no further change
in absorbance was seen by electric stimulation, epinephrine infusion, or nitrogen flowing on the heart
surface. When perfusion pressure was decreased
from 80 to 30 cm H2O, the flow rate and left
ventricular pressure decreased markedly. The oxygen concentration in the effluent perfusate decreased from 0.83 to 0.55 mM, and about 50% of
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Circulation Research/Vol. 53, No. 4, October 1983
(B)
(A)
Calibration
Perfusion Pressure (cmhbO)
60
|30|50| 80 1110 11301 150 11301110'
O2 Concentration
in Effluent
Myoqlotrin I
Oxygenation
(580-620nm)
Heart Rate
Flow Rate
.•D00
|0 snHg
-1000
LVdP/dt
L
-v-
V P
—|5mmh-
-H5mmH-
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FIGURE 1. Various cardiac performances modified externally. Rat
heart was perfused with a Krebs-Ringer bicarbonate solution equilibrated with 95% 02 + 5% C02. Part A: perfusion pressure (shown in
top column) was changed by changing the height between the aorta
and reservoir. Part B: calibration traces were obtained from normoxicanoxic transition, in which the perfusate was switched to that equilibrated with 95% N2 + 5% CO2.
total myoglobin was in the deoxygenated form at 30
cm H2O. Then, the intracellular oxygen concentration was estimated at 4 FIM. Increasing perfusion
pressure up to 50 cm H2O caused increases in left
ventricular pressure and flow rate, whereas the oxygen concentration in the effluent perfusate decreased to 0.22 mM. The original level of myoglobin
oxygenation was nearly restored. Increasing the perfusion pressure stepwise up to 150 cm H2O were
accompanied by parallel increases in left ventricular
pressure, flow rate and the oxygen concentration in
the effluent perfusate. Myoglobin oxygenation was
saturated at 110 cm H2O of perfusion pressure.
Heart rate remained almost constant during the
experiment. The first derivative of left ventricular
pressure (dP/dt) also increased with left ventricular
pressure. WTien the perfusion pressure was increased from 80 to 200 cm H2O, lactate release from
the heart increased from 0.15 ± 0.05 to 0.23 ± 0.02
^mol/min per g wet weight, and the intensity of
pyridine nucleotide fluorescence decreased slightly.
Oxygen consumption increased from 2.5 ± 0 . 1 to
10.1 ± 0.3 Mmol/min per g wet weight with the
increase in perfusion pressure from 50 to 200 cm
H2O, which was linearly related to the cardiac work
(cf. Fig. 5). This agreed with the result of Kobayashi
and Neely (1979), who observed similar linearity
upon a change in the aortic resistance. Since myoglobin was almost fully oxygenated under the conditions studied, it was evident that oxygen supply
did not limit oxygen consumption. The linear dependence of oxygen consumption on cardiac work
was also obtained when heart rate was raised from
90 to 210/min by pacing under 80 cm H2O of
perfusion pressure (data not shown).
Normoxic-Anoxic Transition
When the oxygen-saturated perfusate was
switched to the nitrogen-saturated one (Fig. 2A), the
deoxygenation of myoglobin was completed within
1 minute. The reduction of pyridine nucleotide
started when about 30% of total myoglobin was
deoxygenated. The lactate concentration in the effluent perfusate increased markedly under the hypoxic conditions. After anaerobiosis was achieved,
the nitrogen-saturated perfusate was switched to the
oxygen-saturated one. The oxygenation of myoglobin responded first; then, the oxygen concentration
in the effluent perfusate increased. Pyridine nucleotide started to oxidize when more than 10% of
myoglobin was oxygenated, and reached the original
level after myoglobin was completely oxygenated.
Recovery of the lactate concentration in the effluent
perfusate to the original level was later than the
recovery of myoglobin oxygenation. In comparison
with the experiment in Figure 2A, the oxygen concentration in the inflowing perfusate was changed
at a 10 times slower rate, in Figure 2B, by continuously changing the oxygen:nitrogen ratio in the perfusate. The oxygen dependence of cardiac performances was the same in the two cases when the
oxygen concentration was decreased, but was not
when it was increased from anoxia. Reoxidation of
pyridine nucleotide and recovery of lactate release
were completed before the complete oxygenation of
myoglobin.
Figure 3A shows changes in lactate release, pyri-
-H 5min KFIGURE 2. Changes in various performances of perfused heart due to
normoxic-anoxic transition. Part A. the perfusate equilibrated with
95% 02 + 5% C02 was switched to that equilibrated with 95% N2
+ 5% C02 by using an electric solenoid valve. The perfusate was
changed within 5 seconds. Part B: the heart was perfused first with
95% 02 + 5% C02. The oxygen concentration of inflowing perfusate
was decreased at a rate of 50 IIM O2/min by decreasing the Oj.N2
ratio of the gas mixture, and was again increased from an arrow at a
rate of 100 p.u O2/min.
Araki et a/./Myocardial Tissue Oxygenation
451
oxygen consumption were about 1, 3, and 2.6 /XM,
respectively.
The same results as in Figure 3 were given by
transmission spectroscopy in which a thin light pipe
was inserted into left ventricle and the light transmitted through the left ventricular wall was recorded
(Tamura et al., 1978). Thus, it was concluded that
there was no appreciable difference in the oxygenation state of the heart between endo- and epicardium under our perfusion conditions.
0
10
20
30
70
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Intracellutar Chqgtn Concentration (JJM)
FIGURE 3. Relationships of myoglobin oxygenation to pyridine nucleotide fluorescence, lactate release, and oxygen consumption during
normoxic-anoxic transition. The conditions were as described in
Figure 1 Open circles with a vertical line represent mean values of
six hearts, and the lines represent ± SEM. Data were plotted against
the percent oxygenation of myoglobin in part A and against the
intracellular oxygen concentration in part B. In the top of part B, •
represent respective data obtained from another series of experiments.
dine nucleotide fluorescence, and oxygen consumption as a function of the oxygenation state of myoglobin. Lactate release started to increase when about
30% of myoglobin was deoxygenated, and reached
a half maximum at 50% deoxygenation. The halfmaximal reduction of pyridine nucleotide was observed at 70% deoxygenation. Oxygen consumption
changed inversely with lactate release. Identical
curves were obtained by either increasing or decreasing the oxygen concentration in the inflowing
perfusate at a rate below a certain limit. For instance,
the curves started to change when the oxygen concentration was decreased at rates above 90 /iM/
and when it was increased at rates above 40
min. Therefore, we assumed that the steady state is
maintained during the slow changes in the oxygen
concentration, similar to the steady state which can
be assumed in enzyme reactions. In Figure 3B, these
results are replotted as a function of the intracellular
oxygen concentration, with the use of an oxygen
dissociation curve of myoglobin determined previously in the heart (Tamura et al., 1978). Pyridine
nucleotide fluorescence and lactate release started
to increase at oxygen concentrations lower than 6
and 10 tiu, respectively. Lactate release was 6-fold
higher in anoxia (lower than 0.1 ^M oxygen) than in
normoxia. Oxygen consumption started to fall at
oxygen concentrations lower than 10 HM, and the
rate remained constant above the concentration. The
oxygen concentrations required for half-maximal
pyridine nucleotide reduction, lactate release, and
Effects of Epinephrine and Dopamine in Cardiac
Function
Figure 4A shows the effect of epinephrine infusion upon cardiac performance under normoxic perfusion conditions. The infusion increased both left
ventricular pressure and heart rate. The cardiac work
rose from 6300 to 19110 mm Hg/min and the rate
of oxygen consumption from 3.2 ± 0.2 to 6.7 ± 0.3
jimol/min per g wet weight. The increase in oxygen
consumption caused the decrease in the intracellular
oxygen concentration, resulting in deoxygenation of
myoglobin. Maximally, about 60% of myoglobin
was deoxygenated, and then the oxygen concentration in cardiac tissue was estimated at 3 /IM. The
maximum deoxygenation of myoglobin appeared
after cardiac work or oxygen consumption reached
a maximum. Lactate release started to increase when
about 30% of myoblobin was deoxygenated. The
lactate release depended closely on the oxygenated
state of myoglobin. The maximal lactate release (0.5
^mol/min per g wet weight) was placed at the
maximal myoglobin deoxygenation. The results on
norepinephrine infusion were similar to those on
epinephrine (data not shown).
The effect of dopamine infusion was shown in
Figure 4B, where a dose of 100 Mg/ml was used to
give similar values for cardiac work and oxygen
(A)
(A)
Eprephnnt
(cottjl
FIGURE 4. Effects of epinephrine and dopamine infusions on cardiac
performances under normoxic perfusion. Hearts were perfused with a
Krebs-Ringer solution equilibrated with 95% O2 + 5% CO2 at an
aortic perfusion pressure of 80 cm HiO. The infusions of drugs were
continued for 30 seconds, as indicated by bars. Part A- epinephrine (1
lig/ml). Part B: dopamine (100 ng/ml).
Circulation Research/Vol. 53, No. 4, October 1983
452
(A)
(B)
0.6i
5
% .s-0.53 *
\.
0.2
06
ifai.05
§ | 03
T
\
/
I.01
A
0
10
20
30
50
Intracetlular Oxygen Concentration (pM)
a3oi
0
5
10
15
20
25 30
P»* Systolic Presare « Heart Rate
3
(mmHg/min) » 10~
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FIGURE 5. Relationships between cardiac performances modified by infusions of dopamme, epmephnne, and norepinephnne. The conditions were
as described in Figure 4. Part A: oxygen consumption and lactate release were plotted against cardiac work. O, normoxic-anoxic transition (Figs
2 and 3); A, dopamine; O, epinephrine; • , norepinephnne (Fig. 4). Each vertical line represents ± sEMfor six tO, A) or eight (D, M) hearts. Part
B: lactate release was plotted against the intracellular oxygen concentration. Each vertical line for epinephnne (O) and dopamme (O) represents
± SEMfor eight hearts. The data for norepinephrine (M) were the mean value for two hearts. Solid line was replotted from Figure 3B (middle).
consumption, as compared with epinephrine infusion. Dopamine caused marked increases in left
ventricular pressure, heart rate, and oxygen consumption (maximum; 7.1 ± 0.6 ^mol/min per g wet
weight). Cardiac work rose from 7,000 to 16,800
(maximum) mm Hg/min, accompanying a parallel
increase in oxygen consumption (cf. Fig. 5). Lactate
release did not increase under this condition. The
maximal deoxygenation of myoglobin was about
30% of that obtained with epinephrine. Flow rate
was increased by dopamine but not by epinephrine.
This might explain the difference in myoglobin oxygenation between the two cases.
The relationships between cardiac work, oxygen
consumption, and lactate release were summarized
in Figure 5. Figure 5A shows that the increase in
oxygen consumption depended linearly on the cardiac work when the change was caused either by
dopamine or by an increase in perfusion pressure
(Fig. 1). In contrast, the epinephrine- and norepinephrine-induced increases in oxygen consumption
deviated from the straight line above 5.5 /imol/min
per g wet weight. Lactate release was also plotted
against cardiac work. When cardiac work was varied
from 4,000 to 20,000 mm Hg/min by dopamine or
perfusion pressure, lactate release increased slightly
from 0.15 ± 0.05 to 0.23 ± 0.02 ^mol/min per g
wet weight. At cardiac works above 12,000 mm Hg/
min, lactate release induced by either epinephrine
or norepinephrine increased markedly and deviated
from the plots obtained by changing perfusion pressure.
Lactate release measured under various conditions
was plotted against the intracellular oxygen concentration in Figure 5B. The plots obtained with epinephrine and dopamine all coincided with the curve
obtained by normoxic-anoxic transition shown in
Figure 3B (middle). Changing the dose of epinephrine from 0.01 to 2 ng/m\ did not change the curve
shown in Figure 5 (data not shown).
Fluorescence Changes of Pyridine Nucleotide
Figure 6 shows the effect of increasing oxygen
consumption on the redox state of pyridine nucleotide under normoxic and hypoxic conditions. Increasing heart rate from 90 to 110 in normoxia
caused an increase in oxygen consumption from 2.5
to 3.1 ^mol/min per g wet weight (Fig. 6A). About
30% of myoglobin was then in the deoxygenated
form. Pyridine nucleotide was oxidized and the reduced form was about 10% of that at transient
anoxia. Under hypoxic conditions (Fig. 6B), however, the result was different. At an oxygen concen(A)
(h Concentration
n Effluent
~
Myoglotun
Oxvqenation
(580-620nm|
oxidation of
Pynkie Nucleoti
1360— WOnm)
Flow Rate
FIGURE 6. Effects of heart rate on the redox state of pyridine nucleotide
under normoxic (part A) and hypoxic (part B) conditions. Heart was
perfused with 95% O2 + 5% CO2 in part A and with 40% O2 + 55%
Ni + 5% CO-i in part B. The heart rate rose spontaneously from 90
to no/min by electric pulse.
Araki et a/./Myocardial Tissue Oxygenation
453
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tration of 0.4 mM in the inflowing perfusate, 50% of
myoglobin was deoxygenated. Increasing heart rate
under this condition caused increases in oxygen
consumption from 1.9 to 2.1 ^mol/min per g wet
weight and in myoglobin deoxygenation from 50%
to 65%, similarly as shown in Figure 6A. In contrast
to the result in Figure 6A, however, pyridine nucleotide was further reduced when heart rate increased (Fig. 6B). Upon an increase in oxygen consumption, pyridine nucleotide was oxidized at oxygen concentrations above 10 ^M, but was reduced
below it.
Figure 7 shows redox changes of pyridine nucleotide induced by epinephrine (A) and dopamine (B).
Experimental conditions were the same as those in
Figure 4. The response of fluorescence intensity of
pyridine nucleotide to an epinephrine dose was
triphasic. The initial oxidation of pyridine nucleotide
began with an increase in cardiac work or oxygen
consumption. The switch from oxidation to reduction occurred when 40% of myoglobin was deoxygenated. The maximal reduction of pyridine nucleotide was seen later than maximal deoxygenation of
myoglobin. Re-oxidation of pyridine nucleotide occurred and its redox state reached the initial level
before myoglobin oxygenation and oxygen consumption returned to their initial levels. Thus, lactate
release did not parallel the redox level of pyridine
nucleotide. Infusion of dopamine, on the other
hand, resulted in the oxidation of pyridine nucleotide by 10-15% of the normoxic-anoxic response.
No reduction was seen in this case.
Discussion
Oxygenation State of Myoglobin in the Heart
Under normal perfusion conditions at 27-30°C,
more than 90% of myoglobin is oxygenated (Tamura
et al., 1978) and the intracellular oxygen concentration is kept higher than 50 HM. This is consistent
OjConcentraton
«i Effluent
ImM]
Oxidation of
|
Pyndinc Nucltohdi
(360—WOraa) [
1min
I Smin I
I Sm
FIGURE 7. Effects of infusions of epinephrine (part A) and dopamine
(part B) on the redox state of pyridine nucleotide. The calibration for
normoxic-anoxic changes in myoglobin oxygenation and pyndine
nucleotide fluorescence is shown on the left. Epinephrine (1 iig/ml)
and dopamine (100 ng/ml) were infused for 30 seconds. The other
experimental conditions were as described in Figure 4.
with the observation that absorbance of myoglobin
does not change until the oxygen concentration in
the inflowing perfusate is decreased to 0.7 rrtM (70%
O2 saturation). At a perfusion temperature of 37°C,
however, more than 30% of myoglobin was deoxygenated, even under normoxic perfusion conditions,
where the heart beats spontaneously at a rate of
240-280/min. The intracellular oxygen concentration approaches 10 MM under these conditions. In
order to avoid hypoxia under standard conditions,
the present experiments have been carried out at
27°C. Therefore, the rates of oxygen consumption
and lactate release are slow as compared with those
reported previously (Williamson, 1966a, 1966b;
Neely et al., 1967; Kobayashi and Neely, 1979).
The oxygen concentration in the effluent perfusate (corresponding to the oxygen concentration at
coronary sinus), cannot be used simply as an indicator for the tissue oxygenation state. As seen in
Figure 1, the ratio of oxygen concentrations in the
tissue and in the effluent perfusate is inverted between perfusion pressures of 30 and 50 cm H2O.
Oxygen appears to be transferred from the circulating system into the intracellular space about 10-fold
faster at 50 cm H2O than at 30 cm H2O. This
phenomenon cannot be explained simply by an
increase in the flow rate. Probably, the capillary bed
might close at a perfusion pressure of 30 cm H2O.
Although the oxygen concentration in the effluent
perfusate decreases to about 0.2 mM upon infusion
of either dopamine or epinephrine, the intracellular
oxygen concentration is much higher in the former
than in the latter (Fig. 6). This indicates that an
increased flow rate by dopamine delivers oxygen
into the tissue effectively. Thus, there seems no
parallel between the flow rate, the oxygen concentration in the effluent perfusate, and the tissue oxygenation state. Infusion of adenosine to the perfused heart causes increases both in flow rate and
oxygen concentration in the effluent, whereas
myoglobin is deoxygenated. Dipyridamole gives
similar results (Tamura et al., 1983).
Effect of Oxygen Concentration on Respiration
Rate, Lactate Production, and Pyridine
Nucleotide Fluorescence
The rate of oxygen consumption begins to decrease at an oxygen concentration of 10 HM, which
may be called the critical oxygen concentration in
the heart, although there is an argument with regard
to this terminology (Wilson et al., 1973). This value
is 10-fold higher than that obtained with isolated
mitochondria (Longmuir, 1957; Jobsis, 1972). The
difference can be accounted for by the oxygen concentration gradient between the cytosolic and mitochondrial spaces (Tamura et al., 1978). It is of interest to note that the deoxygenation of myoglobin
commences at the critical oxygen concentration. This
implies that myoglobin participates in regulating the
oxygen concentration in the heart.
Nishiki et al. (1979) have examined the interde-
Circulation Research/Vol. 53, No. 4, October 1983
454
_ 0.5
a
|
0.4
•S * 0.3-£j
0.2
|1 020
1
2
AOxygen Consumption
(>m>oles/ming wet weight)
FIGURE 8. Plots of the extra release of lactate against the deviation
of oxygen consumption from the requirement for work. The deviation
was caused by anoxia (O) and by infusions of epinephrine (O) and
norepinephrine (U). The amounts of deviation correspond to the
difference between the two lines shown in Figure 5A. Vertical and
horizontal lines represent ± SEM.
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pendence of oxygen consumption and glycolytic flux
in the aerobic steady state using Amytal as a respiratory inhibitor, and have shown that time courses
of oxygen consumption and lactate production are
mirror images of one another. The same relation
between oxygen consumption and lactate release is
seen in the normoxic to hypoxic transition (Fig. 3B).
Figure 8 shows that the deviation from the expected
values occurs proportionally with a value of about
5 for —A[O2]/A[lactate]. This relationship might be
applied to blood-circulated heart or skeletal tissues,
in which the lactate level of venous blood has been
used as an indicator for hypoxia.
The fluorescence intensity of pyridine nucleotide
also reflects the intracellular oxygen concentration
(Fig. 3). Since percent reduction of cytochrome a +
a3 is proportional to the oxygenation state of myoglobin (Tamura et al., 1978), the percent reduction
of pyridine nucieotide is plotted against percent
reduction of cytochrome a + a3 in Figure 9. The
curve for the heart resembles that in state 3 of
isolated mitochondria (Sugano et al., 1974; Oshino
et al., 1974).
Chance (1959) and Jobsis et al. (1963), using perfused frog sartorius muscle, have observed that pyridine nucleotide oxidized during contraction is reduced back to the original level in the resting state.
Such a stimulation-induced oxidation of pyridine
nucleotide is seen in normoxia (Fig. 6A). In isolated
mitochondria, an ADP-induced stimulation of respiration causes oxidation of pyridine nucleotide
(Chance et al., 1964). In this connection, Chance
(1964) demonstrated that increased stimulation
caused first an oxidation and then the reduction of
pyridine nucleotide on the frog sartorius muscle.
The reduction of the pyridine nucleotide was explained by the local hypoxia, where oxygen demand
exceeded the supply. In Figure 6B, however, when
the heart was stimulated, the intracellular oxygen
concentration decreased only slightly from 4 to 3.5
JTM, although deoxygenation of myoglobin was
much more distinct. Therefore, a stimulation-induced reduction observed in Figure 6B cannot be
explained clearly. Sugano et al. (1974) have reported
that the plot of pyridine nucleotide reduction against
the oxygen concentration is shifted toward the
higher oxygen concentration when respiration is
increased. For instance, its half reduction is observed
at 0.08 ^M in state 3 and 0.02 JIM in state 4 of
mitochondria. Thus, the stimulation-induced reduction (Fig. 6B) may be accounted for not only by the
development of hypoxia, but also by a shift in the
curve on the formation of ADP from ATP under
hypoxic conditions. It seems likely that the change
in fluorescence intensity comes mainly from the
redox change of pyridine nucleotide in mitochondria.
Epinephrine and Dopamine Infusions
As shown in Figure 5A, on epinephrine infusion,
the rate of oxygen consumption reaches a maximum
(6 Mmol/min per g wet weight), even though the
cardiac work increases further. The further increase
in the cardiac work is accompanied by an increase
in lactate release. The insufficiency of ATP production through oxidative phosphorylation may be
compensated by an increase in glycolytic flux. As
shown by Kobayashi and Neely (1979), and also in
Figure 5A, glycolytic flux was increased when cardiac work was raised in the normoxic conditions.
However, the epinephrine-induced increase of the
flux could not be explained by the above, because
at the same cardiac work, lactate release was more
than 3-fold larger in the heart after epinephrineinfusion than that of increasing perfusion pressure
or dopamine-infusion. Figure 5B shows that such an
increase in the glycolytic flux is rather indicative of
the hypoxic condition in cardiac tissue. The increased amount of lactate release is proprononal to
the decreased amount of oxygen consumption, but
a molar ratio Alactate:Aoxygen of about 0.2 (Fig. 8)
is too small to supplement the lack of ATP production through glycolytic flux. The reason remains to
be determined by further experiments.
Chance et al. (1965) have reported complex re-
0 20 40 60 80 100
% Oxidation of Cyt a +
FIGURE 9. The relationship between the redox changes of pyridine
nucleotide and cytochrome a + a3 in the heart and in isolated
mitochondria. The plots (O) were calculated from data in Figure 3A
and by others (Oshino el al., 1974; Tamura et al., 1978). The solid
line was obtained by Sugano et al. (1974) in state 3 of isolated
mitochondria with glutamate and the dashed line in state 4.
Araki et a/./Myocardial Tissue Oxygenation
sponses of pyridine nucleotide fluorescence to epinephrine. A triphasic response of the fluorescence
seen in Figure 7 A agrees with the result of Williamson (1964), who has interpreted the abnormality as
follows: the initial oxidation corresponds mainly to
oxidation of mitochondrial pyridine nucleotide
through an increase in ADP, and the subsequent
reduction is ascribed to reduction of pyridine nucleotide due to glycogenolysis and glycolysis (Williamson and Jamieson, 1966). Figure 7 A shows that
the reduction of pyridine nucleotide commences
when about 40% of myoglobin is deoxygenated.
Then, the intracellular oxygen concentration is about
8 HM. This concentration appears to be critical to
determine the direction of oxidation-reduction of
pyridine nucleotide in our perfused rat heart
(Fig. 6).
Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017
This study was supported by Grant in Aid for Scientific Research
(343029, 57440097) from the Ministry of Education, Science, and
Culture of japan.
Address for reprints: Mamoru Tamura, Biophysics Division, Research Institute of Applied Electricity, Sapporo 060, Japan.
Received November 2, 1982; accepted for publication July 20,
1983.
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INDEX TERMS: Myoglobin • Hypoxia • Intracellular oxygen
concentration • Critical oxygen concentration
The effect of intracellular oxygen concentration on lactate release, pyridine nucleotide
reduction, and respiration rate in the rat cardiac tissue.
R Araki, M Tamura and I Yamazaki
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Circ Res. 1983;53:448-455
doi: 10.1161/01.RES.53.4.448
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