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JOURNAL OF EXPERIMENTAL ZOOLOGY 283:501–509 (1999)
O2 Consumption and Metabolic Activities in Resting
Cardiac Myocytes From Rainbow Trout
B. MORTENSEN AND H. GESSER*
Institute of Biological Sciences, Department of Zoophysiology, University of
Aarhus DK-8000, Aarhus C, Denmark
ABSTRACT
In mammalian cardiac tissue, the O 2 consumption during mechanical rest is strikingly high. As this may relate to endothermy, the O2 consumption of resting atrial myocytes from
an ectotherm, the rainbow trout, was examined. It was 1.46 ± 0.13, 1.96 ± 0.33, 4.34 ± 0.47, and
6.37 ± 0.79 nmol min–1 mg protein–1 at 5, 10, 15, and 20°C respectively. These values are about one
fourth of the maximal O2 consumption recorded with 30 µM of the uncoupler dinitrophenol. Inhibition of mitochondrial ATP synthetase with 5 µg ml–1 oligomycin reduced resting O2 consumption
by 33 ± 9, 33 ± 1, and 24 ± 7% at 10, 15, and 20°C, respectively. Thus, only about one third of
resting O2 consumption supports ATP synthesis, whereas the rest probably represents a futile
proton cycle across the mitochondrial membrane. Thus, oligomycin-insensitive O2 consumption
was not diminished by 30 µM ruthenium red, which inhibits mitochondrial Ca2+ uptake. About
one half of the 30% of the O2 consumption coupled to ATP synthesis supports Na-K ATPase, inasmuch as 2 mM ouabain in the absence of extracellular Ca2+ reduced O2 consumption by about 15%
at 10 and 15°C and somewhat less at 20°C. With 1.26 mM Ca2+, the reduction disappeared, probably due to the Na-Ca exchange. As examined at 15°C, changes in extracellular Ca2+, i.e., Ca2+
load, from 0 to 1.26 and 7 mM did not affect O2 consumption. Despite an apparent respiratory
“overcapacity,” lactate measurements indicate that between 10 and 20% of the ATP synthesis in
the resting cells is anaerobic. Inhibition by ouabain suggests that about 30% of the lactate production supports Na+-K+-ATPase. J. Exp. Zool. 283:501–509, 1999. © 1999 Wiley-Liss, Inc.
In mammals, the fraction of cellular respiration remaining after cessation of contractile activity is higher in cardiac muscle than in other
muscles. This resting respiration supports ATP
requiring processes including transport ATPases
involved in the cellular regulation of ions such as
Na+, K+, and Ca2+ (Gibbs, ’78; Chapman et al., ’82).
The main part of the resting respiration, however,
does not seem to be coupled to ATP synthesis
(Challoner, ’68; Haworth et al., ’83; MorenoSanchez and Hansford, ’88). Instead, it may directly support mitochondrial transport of ions such
as Ca2+ (McCormack et al., ’90). In addition, a futile proton leak across the inner mitochondrial
membrane seems to be generally present in tissues of mammals (Brand et al., ’94) and have also
been recorded in one ectothermic species, a reptile, at 37°C (Brand et al., ’91).
The futile proton leak appears to participate in
temperature regulation of endothermic vertebrates. For instance, oligomycin-insensitive respiration of mitochondria from heart and skeletal
muscle of the ground squirrel is increased during
winter (Brustovetsky et al., ’92). Furthermore, proton leak has been suggested to fulfill three other
© 1999 WILEY-LISS, INC.
functions. First, it enables aerobic energy liberation to follow rapid changes in energy demand.
Secondly, it may counteract formation of harmful
free radicals. Thirdly, it may regulate different carbon fluxes (Rolfe and Brand, ’97). The first function should be important in cardiac muscle with
respect to its rapid and large variations in contractile activity and its almost exclusive dependence on aerobic energy liberation. In contrast,
activity changes in skeletal muscle typically rely
heavily on anaerobic glycolysis. If the high cellular respiration recorded for resting cardiac muscle
from mammals represents a means for rapid
changes between different activity levels and also
for removal of free radicals and for regulation of
carbon fluxes, it should not only occur in endothermic species but also in ectothermic species at
their different in vivo temperatures.
The present study of rainbow trout concerns the
components of resting myocardial respiration at
Grant sponsor: Danish Natural Science Research Council; ‘‘Center
for Respiratory Adaptation.’’
*Correspondence to: H. Gesser, Institute of Biological Sciences, Department of Zoophysiology, University of Aarhus DK-8000, Aarhus
C, Denmark. E-mail: hans.gesser@biology.aau.dk
Received 15 April 1998; Accepted 11 August 1998.
502
B. MORTENSEN AND H. GESSER
different temperatures within the in vivo range.
In addition to components not associated with ATP
synthesis, the interest was directed to the ionic
regulation depending directly or indirectly on NaK-ATPase, which presumably constitutes a major
part of the energy demand in resting muscle. The
contribution of these different processes to cellular respiration was estimated with various agonists and antagonists. In order to reduce diffusion
limitations, the experiments were performed on
suspensions of cells isolated from atrial tissue of
rainbow trout. Calorimetric studies show that suspensions of cardiac myocytes from mammals are
well suited for metabolic studies like the present
(Ponce-Hornos et al., ’90). This seems to be true
for trout cardiac myocytes as well, according to,
among other things, their high ability to maintain cellular energy state (Milligan, ’91).
MATERIAL AND METHODS
Rainbow trout (Oncorynchus mykiss) weighing
250–300 g were kept in fresh water tanks at 15 ±
2°C and fed regularly. The solutions used in the
different procedures were based on solution A containing (mM): 100 NaCl, 2.5 KCl, 1.8 MgCl2, 0.63
NaH2P04, 10 HEPES and as substrates 5 of each
glucose, Na-glutamate, Na-fumarate, and Na-pyruvate. Unless otherwise stated, solution A also contained 10 mM 2-3-butanedione monoxime (BDM)
in an attempt to obtain a well-defined resting state.
BDM inhibits the interaction of actin and myosin
and the action potential dependent influx of Ca2+
into the cell (Backx et al., ’94, Ikenouchi et al.,
’94). Furthermore, BDM has been shown to counteract dissection injury (Mulieri et al., ’89). At both
10 and 20°C, 10 mM BDM inhibits about 60% of
the twitch force developed by isolated ventricular
strips of trout. The concentration providing full inhibition amounted to more than 30 mM (unpublished observations) and was not chosen because
of a concern about possible side effects. Immediately before use and after its pH had been brought
to 7.4 (20°C) with imidazol, solution A was equilibrated with pure O2 followed by the addition of 1
mg/ml bovine serum albumin.
Atrial cells were isolated enzymatically on the
basis of methods previously used (e.g., Mitra and
Morad, ’85). The thin walled atrium was used to
favor a homogenous enzyme treatment. The fish
was decapitated and the heart excised and transferred to an ice-cold solution A with 250 IU/ml
heparin to inhibit blood coagulation. A plastic tube
was inserted in bulbus and 5 ml of solution A with
heparin were injected to remove blood from the
ventricular and atrial chambers. To isolate atrial
cells, the heart was perfused at a rate of 4 ml sec–1
with 50 ml of solution A and then by 20 ml of solution A to which had been added 1 mg/ml collagenase (0.15 IU/ml) (Boehringer Mannheim) and 0.5
mg/ml (about 5,000 BEE units/ml) trypsin (Sigma,
St. Louis, MO). The latter solution was recirculated
for 30 min. Care was taken to remove air bubbles
and to ensure that the atrium wall was extended
to secure access of the solution to the cells. After
the perfusion, which was performed at 20°C, the
heart was placed on an ice-cold petri dish and the
atrium was cut free. Atrial cells were suspended in
3–4 ml of solution A with 1.25 mM CaCl2. After being spun down for 1 min at 100g, cells were resuspended in 3–4 ml of A with 1.25 mM CaCl2 and
stored at 4°C until being used for recordings of O2consumption or lactate production. The cells were
used the day of preparation in some introductory
experiments to establish suitable protocols. For the
rest they were stored overnight.
The quality of the cells was evaluated immediately before and after recording of respiration or
lactate production. The number of intact and defective cells was determined at a 10-times magnification in a hematocytometer by counting cells
in four squares containing 8 · 10–9 liters. The cells
should be long (about 150 µm) and thin (about 10
µm) and have a smooth surface. The preparations
contained two types of defective cells of which one
was round. According to literature, these cells are
muscle cells in a supercontracted state although
the possibility that also non-muscle cells are included in this group cannot be excluded. The metabolic activity of supercontracted muscle cells is
unclear. According to measurements of heat development in suspensions of cardiac myocytes
from rat, guinea pig, and rabbit, supercontracted
cells do not seem to contribute significantly to
metabolic activity (Ponce-Hornos et al., ’90),
whereas their activity in terms of oxygen consumption was somewhat above normal for turtle
cardiac myocytes (Watson et al., ’94). The other
type of defective cells was long and slender but
with blebs on the cell membrane, probably indicating a disturbed ion regulation (Watson et al.,
’94, Uchida and Doi, ’94). The preparations of the
present study contained up to 15% abnormal cells,
of which maximally 5% were supercontracted and
the rest were long and slender but with blebs. The
dye tryptan blue, which was used to test the barrier function of the cell membrane (e.g., Watson
et al., ’94), entered supercontracted cells but was
excluded from all the slender cells irrespective of
O2 CONSUMPTION OF TROUT MYOCYTES
the presence of blebs. The quality of the preparations was also examined at room temperature
(about 20°C) by incubating about 20 cells in 250
µl solution with or without BDM. Irrespective of
the presence of BDM, all of the slender cells without blebs were quiescent, whereas the majority of
those with blebs displayed spontaneous contractions. This spontaneous activity was not assessed
quantitatively, because the activity varied between
cells as well as for the individual cell. Thus, cascades of spontaneous contractions of a frequency
up to about 1 Hz alternated with periods of rest.
In the absence of BDM, all of the slender cells
could be stimulated to contraction by electrical
square pulses of a duration of 5 msec delivered
by a stimulator (Grass SD 9, Quincy, MA) through
two platinum electrodes. Cell quality was influenced by the experimental temperature. Thus
most of the cells became blebbed and spontaneously active at 20°C, whereas the temperatures
below 20°C did not affect cell quality.
•
Cellular O2 consumption (MO2), referring either to mg cellular protein or to ml cell suspension, was recorded polarographically by an
electrode (Radiometer E5046) in a thermostated
1.4 ml glass vessel filled with suspension, which
was stirred by a magnetic stirrer. With intervals of five seconds, O2 tension was recorded
and stored on computer. The initial O2 tension
was 250–300 mmHg and the final one• not below 150 mmHg. In this tension range, MO2 was
not limited by oxygen availability. This was
demonstrated
in an experiment run at 15°C in
•
which MO2 was stable, although oxygen tension
fell from 150 to 120 mmHg and respiration was
stimulated by 30 µM of the uncoupler dinitrophenol (Fig. 1).
Lactate was measured spectrophotometrically
as NADH produced by the LDH reaction coupled
to the glutamate-pyruvate transaminase reaction
(Boehringer, Mannheim). Values of O2-consumption and lactate production were related to tissue
protein determined as described by Peterson (’77).
The cell sample had been incubated at the experimental temperature for at least 10 min before recordings of respiration or lactate production
were started. Recordings were then performed for
15–20 min before any experimental intervention
was applied.
Values are presented as mean ± SEM with the
number of measurements in parenthesis. Significance was assessed with ANOVA and Student test
for paired and unpaired data. Arcsin transformation was performed for normalized data (Sokal and
503
Fig. 1. Decrease in pO2 with time at 15°C in a suspension
•
of myocytes; MO2 was stimulated with 30 µM of the uncoupler
•
dinitrophenol. MO2 = 7.8 nmol O2 min–1 ml suspension–1.
Rohlf, ’81). Significance was considered present
at P < 0.05.
RESULTS
•
MO2 of resting myocytes increased with temperature, although the difference between 5 and
10°C. was not statistically significant (Table 1).
Due to this fact, 5°C was not included in the further experiments. According to experiments per•
formed at 15°C, the MO2 recorded is exclusively
due to the mitochondrial respiratory chain being
completely inhibited by either 2 mM NaCN (n =
2) or 12.5 µM rotenone (n = 2).
•
In order to estimate its maximum, MO2 was recorded in the presence of 30 µM of the uncoupler
•
dinitrophenol, DNP. MO2 increased several times
by a percentage that did not differ significantly
among the temperatures applied (Table 2A). Preliminary experiments at 10 and 20°C had shown
that DNP elicited its maximal effect at 30 µM, as
•
MO2 did not change upon an elevation of DNP
from 30 to 60 µM (data not shown).
Oligomycin—a well-established inhibitor of
mitochondrial ATP synthetase—was applied to
•
to ATP
estimate the fraction of MO2 coupled
•
synthesis. With 5 µM oligomycin, MO2 was lowered by about one third and that, to an extent,
did not vary significantly among the temperatures applied (Table 2B). The maximal effect of
•
oligomycin on MO2 was developed at 5 µmol liter–1 according •to experiments run at 10 and
20°C, in which MO2 was measured subsequently
at 5 and 15 µmol liter–1 oligomycin. The reduc•
tion in MO2 after the first and second addition
504
B. MORTENSEN AND H. GESSER
•
TABLE 1. MO2 (nmol min–1 mg protein–1) and temperature1
°C
•
M O2
5
10
15
20
1.46 ± 13 (9)
1.96 ± 0.33 (5)
NS2
4.34 ± 0.47 (7)
P < 0.01
6.37 ± 0.79 (7)
P < 0.05
•
1
Mean values ± S.E.M. Number of samples within parentheses. The significance levels refer to the difference in MO2 between the temperature indicated and that which is 5°C lower.
2
NS = nonsignificant.
of oligomycin respectively was 34 ± 4% and 33
± 10% (n = 3) at 10°C and 29 ± 5 and 26 ± 7 (n
= 3) at 20°C. Oligomycin was dissolved in
•
DMSO, which did not affect MO2 when applied
in concentrations used in the present experiments, i.e., less than 2% of the total volume.
Oligomycin should block mitochondrial ATP
synthetase completely and should therefore remove the influence of an increased ATP demand
•
on MO2. This was tested with veratridine, which
is known to stimulate energy degradation of
myocardial cells by increasing the cytoplasmic
Ca2+ activity and the ATP hydrolysis of the contractile system (Moreno-Sanchez and Hansford,
’88). In experiments performed at 10 and 15°C,
•
MO2 was measured in a solution, from which
BDM and its inhibition of the ATP hydrolysis
by the contractile system were excluded. Without
oligomycin, 30 µM veratridine stimulated
•
M O 2 to an extent, which was significantly
higher at 15 than 10°C (Table 2C1). When veratridine was added 20 min after oligomycin had
•
TABLE 2. MO2 (nmol min–1 ml suspension–1) recorded just before the addition of the agent indicated,
•
and change (%) in MO2 after stabilization in presence of the agent1
°C
10
A. Dinitrophenol (DNP), 30 µmol liter
•
MO2
1.00
DNP %
435
B. Oligomycin, 5 µg ml–1
•
MO2
1.42
oligom %
–32
C1. Veratridine, 30 µmol liter–1, no BDM
•
1.23
MO2
veratridine %
59
15
20
–1
± 0.12 (11)
± 57 P < 0.001
1.97 ± 0.30 (11)
385 ± 44 P < 0.001
2.58 ± 0.37 (11)
556 ± 62 P < 0.01
± 0.48 (11)
± 10 P < 0.025
2.52 ± 0.30 (11)
–33 ± 1 P < 0.001
4.55 ± 0.79 (11)
–24 ± 7 P < 0.025
5.34 ± 0.73 (4)
200 ± 39 P < 0.025
P < 0.025 (10 vs. 15°C)
C2. Veratridine, 30 µmol liter–1 in the presence of oligomycin, 5 µg ml–1, no BDM
•
1.51 ± 0.46 (7)
2.80 ± 0.52 (8)
MO2, oligom
veratridine %
4 ± 7 NS
1 ± 3 NS
D1. Cyanide (CN), 0.25 mmol liter–1 in the presence of iodoacetate (IAA), 1 mmol liter–1
•
MO2, IAA
2.14 ± 0.70 (6)
CN %
–38 ± 7 P < 0.01
D2. Oligomycin, 5 µg ml–1 in the presence of iodoacetate (IAA) and cyanide (CN)
•
1.43 ± 0.51
MO2, IAA, CN
oligom %
–32 ± 2 P < 0.001
E1. Ouabain, 2 mmol liter–1, with Ca2+, 1.26 mmol liter–1
•
MO2
1.41 ± 0.07 (12)
2.98 ± 0.62 (9)
ouabain %
8 ± 8 NS
2 ± 8 NS
E2. Ouabain, 2 mmol liter–1, without Ca2+
•
MO2, 0 CA2+
2.07 ± 0.33 (12)
3.01 ± 0.59 (9)
ouabain %
–16 ± 4 P < 0.01
–14 ± 4 P < 0.01
F. Extracellular Ca2+ mmol liter–1
•
MO2, 0 Ca2+
2.08 ± 0.96 (10)
–1 ± 8 NS
1.26 Ca2+ %
•
2.01 ± 0.86 (10)
MO2, 1.26 Ca2+
4 ± 8 NS
7 Ca2+ %
G. Ruthenium red, 30 µmol liter–1 with oligomycin, 5 µg ml–1
•
MO2, oligom
1.79 ± 0.40 (5)
Ruth. red %
5 ± 6 NS
1
± 0.34 (4)
± 18 P < 0.05
4.02 ± 0.83 (13)
20 ± 8 P < 0.05
3.49 ± 0.75 (10)
–7 ± 6 NS
The experiments were run at the temperatures given at the top of each column. Unless otherwise stated, significance levels refer to the
effect of the agent. cf. Table 1 footnotes.
O2 CONSUMPTION OF TROUT MYOCYTES
been applied, the stimulation of respiration by
veratridine was removed at both temperatures
(Table 2C2).
The fraction of respiration sensitive to oligomycin has been shown to increase with the rate
of ATP hydrolysis, i.e,. when the energy demand
is increased relative to the energy liberating capacity (Haworth et al., ’83). Therefore, the effect of oligomycin was recorded following an
attempt to increase energy demand relative to
the energy liberating capacity by decreasing the
latter with 0.25 mM NaCN and 1 mM iodoacetate. Cyanide in this concentration decreases
both the electron flow in the respiratory chain
and as a result the proton gradient across the
mitochondrial inner membrane (Brand et al.,
’94). Furthermore, a shift from aerobic to
anaerobic ATP synthesis was prohibited by an
inhibition of glycolysis with iodoacetate (Hartmund and Gesser, ’96). Iodoacetate was added
first followed 10 min later by cyanide. During
the
10 min following the addition of cyanide,
•
MO2 stabilized at a value lower than the initial
one (Table 2D1). The subsequent
addition of oli•
gomycin, 5 µg/ml, reduced MO2 further by a percentage that did not differ significantly from
that recorded in the absence of cyanide and
iodoacetate (Table 2D2).
In cardiac myocytes, a substantial part of the
ATP hydrolysis supports ionic homeostasis, which
to a major extent either directly or indirectly depends on Na+-K+-ATPase (Skou, ’92; Brand et al.,
’94). A survey of the literature covering a wide
range of cell types provides evidence that 2 mmol
liter–1 should give a complete inhibiton of Na+-K+ATPase in trout heart cells. For instance, a K0.5
of 20 µmol liter–1 for ouabain was found with cultured chick cardiac myocytes (Stimers et al., ’90).
–1
ouabain entailed a significant
Two mmol liter
•
elevation of MO2 at 20°C and no significant change
at either 10 or 15°C (Table 2E1). This heterogenous response may depend on the Na+-Ca2+ exchange across the cell membrane allowing Na+ and
Ca2+ to accumulate together in the cell following
an inhibition of Na+-K+-ATPase at a rate that increases with temperature. Therefore, experiments
were performed in the nominal absence of extracellular•Ca2+. Here, a tendency of ouabain to decrease MO2 appeared, which was significant at all
temperatures except 20°C. The effects at the three
temperatures were not statistically different
(Table 2E2).
The different effects of ouabain recorded with
and without extracellular Ca2+ are most probably
505
an expression of the function of Na-K-ATPase acting through the Na-Ca exchange in the maintenance of a low cytoplasmic Ca2+ activity. This
•
function entails the possibility that the MO2 associated with Na-K-ATPase is underestimated,
•
when the decrease in MO2 by ouabain is recorded
in a Ca-free solution. This reasoning implies that
extracellular Ca2+ should enhance the Na-K•
ATPase activity and MO2. Furthermore, extracel•
lular Ca2+ is of interest regarding the part of MO2,
which is insensitive to oligomycin, as mitochondria are able to accumulate Ca2+ by a mechanism
directly supported by respiration (McCormack et
al., ’90). The effect of Ca2+ was examined at 15°C.
In one series of experiments, cells were incubated
in a solution of which Ca2+ was maintained in sequence at 0, 1.26, and 7 mmol liter–1 with 15 min
at each concentration. Neither the
first nor the
•
second elevation of Ca2+ changed MO2 significantly
(Table 2F). The respiratory-supported mechanism
by which mitochondria takes up Ca2+ is inhibited
by ruthenium red (McCormack et al., ’90). Therefore, ruthenium red in a concentration of 30 µM,
which is in the upper range of concentrations used
on whole myocyte preparations (e.g., Siegmund et
al., ’92), was applied in another series of experiments. These experiments were run in the
presence of oligomycin to block the respiration
supporting ATP synthesis and to accentuate the
respiratory dependent part of cellular Ca2+ handling. No significant effect of ruthenium red on
•
MO2 was detected (Table 2G).
Lactate production was assessed in experiments run at 15°C and revealed a significant
anaerobic energy liberation (Table 3). Evidence
exists that transport processes across the cell
membrane preferentially utilize anaerobically
produced energy (Campbell and Paul, ’92).
Therefore, the effect of ouabain on lactate production was examined in a separate series of experiments performed in a Ca2+-free solution to
avoid possible side effects of an increase in cellular Ca2+. The average amount of lactate released
during 20 min fell significantly during the following 20 min with ouabain, whereas no significant change was recorded for the cells subjected
to the same protocol but without ouabain (Table
3). According to the numbers in Table 3, the ouabain inhibited lactate production amounted to 28
± 12% (P < 0.05). The value for the lactate production rate recorded in the presence of extracellular Ca2+ did not differ significantly from the
two values obtained in the absence of extracellular Ca2+ (Table 3).
506
B. MORTENSEN AND H. GESSER
TABLE 3. Lactate production rate (nmol min–1 mg–1 protein) at 15°C in 1.26 and 0 mmol liter–1 Ca2+ and change (%) in
lactate production in 0 Ca2+ after 20 min in presence of either 2 or 0 mmol liter–1 ouabain1
Ca2+ mmol liter–1
126
Lactate, nmol min–1 mg–1
0
0.99 ± 0.16 (13)
20 min, %
1
0.78 ± 0.09 (8)
+Oua
–43 ± 5 P < 0.001
0
0.87 ± 0.10 (8)
–Oua
–15 ± 11 NS
Significance levels refer to the changes in lactate production. cf. Table 1 footnotes.
DISCUSSION
The respiration recorded in the present study
is exclusively mitochondrial in relation to its
inhibition by either cyanide or rotenone. The
temperature dependence of resting respiration
was similar to that seen in other biochemical or
physiological processes having a Q10 well above
one. In a study similar technically to the present
one, ventricular myocytes from turtle displayed
an oxygen consumption of about 1 nmol mg protein–1 min–1 at about 20°C (Watson et al., ’94),
i.e., about six times lower than that recorded for
trout atrial cells at 20°C in the present study.
This difference may relate to differences in cellular metabolic capacities between the two species
(Christensen et al., ’94) and the finding that atrial
tissue is likely to have a higher metabolic activity
than ventricular tissue as judged from its higher
activity of Ca2+ activated myosin ATPase (Degn
and Gesser, ’97).
In this respect, it also should be noted that the
O2 consumption recorded for trout atrial tissue is
well below maximal as it was enhanced 3.5–5.5
times by the uncoupler DNP. Resting respiration
relative to the maximal respiratory capacity estimated by the O2 consumption in the presence of
DNP was not significantly affected by temperature in the range of 10–20°C. It is doubtful, however, if a respiration of the magnitude released
by the DNP ever occurs in vivo. Considering this,
trout atrial cells seem to resemble the mammalian heart in which resting metabolism measured
in terms of O2 consumption or heat production constitutes a substantial fraction of the metabolism
during mechanical activity (Gibbs, ’78; Chapman
et al., ’82). For technical reasons it should be noted
that the cells at 20°C were of lower quality than
those at the other temperatures; almost all had
blebs. The fact that their behavior was reasonably within the range of the other cells seems to
indicate that the presence of cells with blebs did
not seriously disturb the results.
Veratridine was used to examine the efficiency
of the oligomycin inhibition. Evidence exists that
it attaches itself to open sarcolemmal Na+ chan-
nels and potentiates their opening (Horackova and
Vassort, ’74; Honerjäger and Reiter, ’75) so that
there is an intracellular increase in Na+ and subsequently in Ca2+, due to the Na-Ca exchange. The
stimulating effect of veratridine on respiration fell
with decreasing temperature, i.e., from 15 to 10°C.
It is possible that the frequency of spontaneously
opened channels to which veratridine is able to
attach decreases with decreasing temperature.
The removal of the stimulation of respiration by
veratridine and dose-response experiments indicates that the dose of oligomycin used was sufficient to block ATP-synthetase completely. The
effect of oligomycin suggests that about 70% of the
resting O2-consumption of trout atrial myocytes is
not coupled to ATP production. The fraction of respiration coupled to ATP synthetase was not significantly influenced by temperature within the
range covering the in vivo temperatures of rainbow trout. In contrast, the fraction of respiration
coupled to ATP synthetase decreased with lowering of temperature in mitochondria from rat liver
in a way that seemed to depend on adenosine
translocase (Quentin et al., ’94). Regarding the
value of resting O2 consumption not coupled to
ATP synthetase—about 70%—trout myocardium
resembles the myocardium of mammalian, or endothermic, species (Challoner, ’68; Haworth et al.,
’83; Moreno-Sanchez and Hansford, ’88).
The absence of the effect of blocking mitochondrial Ca2+ uptake with ruthenium red in the
presence of oligomycin and of varying the Ca2+
concentrations in the extracellular solution suggests that cellular Ca2+-handling in resting trout
heart cells does not contribute substantially to
cellular respiration. A similar conclusion was
reached for guinea pig heart (Hanley et al., ’94).
Although, other transports such as phosphate and
adenosine (Quentin et al., ’94) were not examined,
it seems probable that the predominant part of
the nonphosphorylating respiration is associated
with a futile proton cycle, i.e., a proton leak. The
size of this respiration in cells from an ectothermic vertebrate and the fact that its fraction of
resting respiration appears to be the same within
O2 CONSUMPTION OF TROUT MYOCYTES
the in vivo range of temperatures complies with
the contention that the futile proton cycle represents a fundamental property of the living cell.
Although its function is unclear, its presence in
cells of ectothermic species suggests that it has
not evolved with endothermy. A similar conclusion
was reached in a comparison of lizard and rat tissues (Brand et al., ’91).
The fraction of respiration not sensitive to oligomycin may represent an overestimation because an
inhibition of ATP-synthetase blocks one pathway of
proton flux across the inner mitochondrial membrane, whereby protons should tend to accumulate
and increase proton motive force. As a result proton flux along the remaining pathways should be
enhanced and respiration should decrease less than
expected from a simple subtraction of the proton
flux coupled to ATP-synthetase (Brand et al., ’94).
However, as oligomycin reduced respiration only by
about 30%, the increase in proton motive force and
the overestimate of the nonphosphorylating part
should be modest. The large fraction of oligomycininsensitive respiration may represent a capacity of
the respiration supporting ATP synthetase to increase promptly upon an increase in cellular ATP
demand (Rolfe and Brand, ’97).
A study of heart cells from rat accords with this
shift from a nonphosphorylating to a phosphorylating respiration in that an enhancement of ATP
degradation increased the fraction of respiration
inhibited by oligomycin (Haworth et al., ’83). On
this basis it is not unexpected that the fraction
of respiration that is insensitive to oligomycin in
noncontracting heart cells would be considerably
higher than in cells such as hepatocytes (Brand
et al., ’94), which may be less removed from their
ATP demand in vivo than noncontracting heart
cells. The results of the present study deviate,
however, from the outlined model inasmuch as
the decrease in proton gradient presumably accompanying the approximate 40% inhibition of
mitochondrial respiration with cyanide did not
change the fraction of respiration insensitive to
oligomycin. The expectation was that the oligomycin-sensitive respiration, i.e., that coupled to
ATP synthetase, should increase and that associated with a proton leak should decrease. This is
because a partial inhibition of mitochondrial respiration should lower proton motive force and total proton flow. Together with an iodoacetate
inhibition of anaerobic ATP synthesis, this should
tend to decrease the cellular energy state and
thus increase the part of the remaining proton
flow coupled to ATP synthetase.
507
A major portion of the phosphorylating respiration—about one third of resting respiration—
inhibited by oligomycin seems to support the
Na+-K+ pump. This portion was not significantly
affected by temperature, such that the energy
degraded by the Na-K pump changed in pace
with total cell respiration. In the resting cell
at steady state, the Na-K pump rate should be
equal to the passive flux of Na+ and K+ across
the membrane. Hence, this flux should have a
Q10 of that typical for enzyme catalyzed processes suggesting that it occurs predominantly
through specific channels. The contribution of
the Na-K ATPase to the energy degradation is
probably underestimated in the present experiments performed in a Ca2+-free solution. Here,
the Na+ load is diminished as Ca2+ does not leak
into the cell and consequently does not have to
be removed by the sarcolemmal Na-Ca exchange
at the cost of an influx of Na+. The importance
of this exchanger was suggested by the increased O2 consumption and the contractures
of the cells, when ouabain was applied in the
presence of extracellular Ca 2+. It should also be
restated, however, that without an increase in
cellular Na+, changes in the cellular Ca2+ load
in terms of extracellular Ca2+ had no substantial influence on O2 consumption. The fraction
of respiration supporting Na-K ATPase might
also be underestimated since an inhibition of
this enzyme may tend to increase the proton
motive force across the inner mitochondrial
membrane and thus increase the resulting proton flux. This effect is likely to be small as the
decrease in respiration due to ouabain only
amounted to about 15%.
Measurements at 15°C suggest that about 10–
20% of ATP is produced by anaerobic metabolism
in assuming a P/O ratio of 3 of the respiration
inhibited by oligomycin and 1–1.5 ATP per lactate. The lactate production of 0.98 nmol min–1
mg–1 protein should be compared to an O2 consumption of about 4.5 nmol min–1 mg–1 protein of
which only about a third represents ATP production according to the oligomycin experiments.
Apart from O2 consumption, ouabain also decreased lactate production by about one fourth,
indicating that the Na+-K+ pump under aerobic
conditions is supported by both aerobic and
anaerobic metabolism. Roughly estimated, the
anaerobic contribution seems to cover one tenth
of the pump activity. The functional advantage of
such an incomplete substrate utilization, i.e., from
glucose and/or glycogen to lactate, is not clear. In
508
B. MORTENSEN AND H. GESSER
the fully aerobic smooth muscle, lactate is produced at a rate that correlates with the Na+-K+
pump activity and it has been proposed that glycolysis offers an advantage due to its cytoplasmic
localization close to the pump sites (Campbell and
Paul, ’92).
In conclusion, the respiration of resting trout
atrial cells constitutes about one fourth of the respiratory capacity and only one third of it appears
to support ATP synthesis, whereas the remaining
two thirds predominantly cover a futile proton
cycle across the inner mitochondrial membrane.
Despite the apparent aerobic overcapacity, a small
but significant fraction of the ATP production is
supported by anaerobic glycolysis. A substantial
part of the phosphorylating aerobic metabolism
as well as of the anaerobic metabolism appears
to support Na-K ATPase. The respiratory rate
changes with a Q10 of about two or above in the
temperature range of 5–20°C, whereas the relative contributions of the processes mentioned essentially stay the same.
ACKNOWLEDGMENTS
The development of the PC-based recording of
data by engineer E. Larsen and the technical assistance of A. Wetter are deeply appreciated.
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