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1 Department of Physiology and Pharmacology, Karolinska Institute, and 2 Department of Sport and Health Science, Stockholm University College of Physical Education and Health, SE-11486 Stockholm, Sweden; and 3 Institute of Chemical Physics and Biophysics, 0026 Tallin, Estonia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested
the hypothesis that the respiratory function of skeletal muscle
mitochondria is impaired by lactic acidosis and elevated concentrations
of Pi. The rate of respiration of chemically skinned fiber bundles from rat soleus muscle was measured at
[Pi] (brackets denote concentration) and pH values
similar to those at rest (3 mM Pi, pH 7.0) and
high-intensity exercise (20 mM Pi, pH 6.6). Respiration was
measured in the absence of ADP and after sequential additions of 0.1 mM
ADP, 20 mM creatine (Cr; 
Cr), and 4 mM ADP.
Respiration at 0.1 mM ADP increased after addition of Cr. However,
Cr was 23% lower (P < 0.05) during
high-intensity conditions than during resting conditions.
Cr was also reduced when Pi or
H+ was increased separately (P < 0.05).
Respiration in the absence of ADP and after additions of 0.1 mM ADP and
4 mM ADP was not affected by changes in [Pi] or
[H+]. The response was similar, irrespective of when
acidosis was induced (i.e., quiescent or actively respiring
mitochondria). In conclusion, Cr-stimulated respiration is impaired by
increases in [H+] and [Pi] corresponding to
those in exercising muscle. Although the reduced Cr-stimulated
respiration could be compensated for by increased [ADP], this might
have implications for intracellular homeostasis.
acidosis; mitochondria; muscle energetics; oxidative phosphorylation; phosphate
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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DURING EXERCISE, THE RATE OF oxidative phosphorylation in skeletal muscle can increase over 100 times (1). The primary signal for the stimulation of respiration, at least in skeletal muscle, is ADP (2, 3). It has been shown that the sensitivity of respiration to ADP is lower (higher Km) in permeabilized (skinned) muscle fibers than in isolated mitochondria. Evidence exists that the ADP permeability of the outer mitochondrial membrane is restricted in skinned fibers (16) but not in isolated mitochondria, in which the structural connections between cytoskeleton and mitochondria are lost during the preparation (16). Increases in creatine concentration ([Cr]) (17) and, as recently shown in our laboratory, decreases in phosphocreatine concentration ([PCr]) (23) stimulate fiber respiration at submaximal but not at maximal [ADP]. The effect of Cr-to-PCr ratio (Cr/PCr) on respiration has been explained on the basis of a "Cr shuttle" (8, 15) by which the [ADP] in the intermembrane space is modulated. Briefly, mitochondrial Cr kinase (CK) (mitCK) is located on the outside of the inner mitochondrial membrane near adenine nucleotide translocase. Porin pores in the outer mitochondrial membrane limit the diffusion of ADP and ATP. Cr and PCr are shuttled between the sites of energy usage (e.g., myofibrils and Ca2+ pumps) and mitochondria (16). During muscle contraction, PCr is broken down, and the increase in Cr/PCr will, through the CK equilibrium, lead to local increases in ADP near adenine nucleotide translocase, which will stimulate respiration. The Cr shuttle can be regarded as an amplifier of the cytosolic ADP signal.
The chemical composition of the working muscle changes drastically during conditions of anaerobic ATP formation because of high rates of glycolysis and PCr depletion. Accumulation of metabolic products will have a strong influence on the rate of the energy-yielding reactions. For example, the activities of key regulatory enzymes of glycolysis (glycogen phosphorylase and phosphofructokinase) are impaired by H+ but stimulated by Pi and AMP. Furthermore, it is well documented that oxidative phosphorylation is stimulated by the products of ATP hydrolysis (ADP and Pi). Product accumulation may, in addition to feedback control of metabolic pathways, disturb the function of cellular processes. The complex nature of oxidative phosphorylation may make this process especially vulnerable to changes in intracellular chemical composition.
In many (4, 5, 7, 9), but not all (11, 18, 19), studies, acidosis has been shown to reduce maximal ADP-stimulated respiration in isolated mitochondria. Acidotic depression of respiration was observed also in skinned cardiac fibers (24). However, the effect of acidosis on respiration in fibers from skeletal muscle is not known. Pi is a prerequisite for oxidative phosphorylation and increases in Pi up to ~5 mM have been shown to increase maximal rate of respiration in isolated mitochondria (6). However, when [Pi] exceeds 5 mM, mitCK dissociates from the inner membrane (10, 25), and high levels of Pi may, therefore, change the control of respiration. The finding that high [Pi] reduces Cr-stimulated respiration in cardiac fibers (24) supports this contention.
During high-intensity (HI) exercise, the metabolic perturbations in skeletal muscle can be large, [Pi] can exceed 20 mM (13), and pH can drop to 6.6 (12). However, the effects of elevated concentrations of Pi and H+ on oxidative function have not previously been studied in skeletal muscle fibers. Therefore, the purpose of this study is to determine the effects of conditions encountered during HI exercise (increased Pi and H+) on the oxidative function of fibers from skeletal muscle.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Subject data. Soleus muscle was obtained from Sprague-Dawley rats weighing 250-300 g (BKI:SD). The rats were housed two to three per cage on 12:12-h light-dark cycles. The animals were given free access to B&K Universal standard rat and mouse diet and tap water. The rats used in this study were cared for in accordance with Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (recommended by the American Association for Laboratory Animal Sciences). The local ethics committee on animal experiments approved the experimental protocol.
Experimental procedure. Animals were injected with 300 µl heparin to prevent clotting and were anesthetized with pentobarbital sodium (100 mg/kg body wt ip). Once the animals were anesthetized, the heart was removed, and the soleus muscles were dissected. The muscle was placed in an ice-cold medium consisting of (in mmol/l) 10 EGTA-Ca-EGTA buffer (free Ca2+ concentration, 100 nM), 20 imidazole, 3 KH2PO4, 0.5 dithiothreitol, 20 taurine, 5.3 ATP, 15 PCr, 9.5 MgCl2, and 53.5 MES, pH 7.00. The fiber bundles were separated with sharp-ended needles, leaving only small areas of contact, and incubated in 1.5 ml of the above medium (4°C) containing 50 µg/ml of saponin for 30 min with mild stirring. To completely remove saponin and metabolites, the fibers were washed three times with mild stirring for 5 min in 1.2 ml of a cooled (4°C) washing and oxygraph medium. Two washing and oxygraph mediums were used in this study to investigate the effect of Pi (3 or 20 mM) on oxidative function. Both mediums consisted of (in mmol/l) 10 EGTA-Ca-EGTA buffer (free Ca2+ concentration, 100 nM), 20 imidazole, 0.5 dithiothreitol, 20 taurine, 4 MgCl2, 74 sucrose, 5 pyruvate, 2 malate, 100 MES, and 2 mg/ml BSA, pH 7.00. Additionally, the washing and oxygraph solutions contained either (in mmol/l) 3 KH2PO4 and 27 HEPES or 20 KH2PO4 and 10 HEPES. The concentration of HEPES was altered to maintain identical osmolarity between the solutions. After washing, the fibers were stored in the washing and oxygraph solution on ice until determination of respiratory activity.
Measurements of mitochondrial respiration.
Mitochondrial oxygen consumption was measured polargraphically with a
Clark-type electrode (Hansatech DW1, Norfolk, UK) in a water-jacketed
glass chamber maintained at 25°C. Measurements were performed in 0.3 ml of the above-described washing and oxygraph solution. In all fibers,
respiration was measured in the absence of ADP (0)
and after the sequential addition of 0.1 mM ADP
(submax), 20 mM Cr (Cr), and 4 mM
ADP (max). Measurements were performed on fiber
bundles from each rat under conditions similar to that at rest
(control: 3 mM Pi, pH 7.0), HI exercise (HI: 20 mM
Pi, pH 6.6), and combinations of control and HI (20 mM
Pi, pH 7.0 and 3 mM Pi, pH 6.6). Acidosis was
induced either before respiratory measurements or after steady-state
submax was attained.
0, submax, and
Cr) was measured in 20 mM Pi (pH 7.0),
washed in oxygraph solution containing 3 mM Pi for 2 min,
and remeasured in the presence of 3 mM Pi. Previously, we
have shown that 15 min of rewashing does not affect ADP-stimulated
respiration (22).
The pH of the solutions was changed to 6.6 through the addition of
lactic acid, to a final concentration of 11 mM. The higher buffer
capacity of the oxygraph solution at 20 mM Pi than at 3 mM
Pi solution was compensated for by additions of HCl.
Additionally, ADP was neutralized with KOH to prevent pH change after
additions of ADP. The average pH of the oxygraph solutions (measured
after each analysis) was 6.56 ± 0.01. Immediately after
respiratory measurements, the fiber bundles were removed, quick-frozen
in liquid nitrogen, freeze-dried, and weighed. Wet weight was used as a
reference base for the respiration and was obtained from the dry
weight, assuming 77% water content.
Data analysis. All values are presented as means ± SE. Differences between means were tested for statistical significance with either a Student's t-test or repeated-measures ANOVA with Student-Newman-Keuls post hoc test. Significance was set at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Pi and pH on respiration.
In accordance with previous findings, respiration in permeabilized
muscle fibers was activated by increases in [ADP] and [Cr] (Fig.
1). During HI conditions (pH 6.6, Pi 20 mM) Cr-stimulated respiration (Cr)
decreased by 23 ± 7% (P < 0.05 vs. control). 0, submax, and
max were unchanged by the increased concentrations of Pi and H+ (Table
1). Therefore, the relative sensitivity
of mitochondrial respiration to ADP [ADP sensitivity,
(submax 
0)/(max 0)] and the respiratory control index
(max/0) were unchanged after treatment. Because of the decrease in Cr-stimulated respiration, mean ADP sensitivity in the presence of Cr
[(Cr 0)/(max 0)] decreased by 40% from 0.53 ± 0.03 to
0.32 ± 0.01 (P < 0.01).
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Cr
and ADP sensitivity in the presence of Cr (Table 1), whereas
0, submax, and
max were not significantly different compared with control.
Influence of respiratory state on the effect of
H+.
Previous studies have shown that the effect of acidosis is dependent on
the functional state of isolated mitochondria at the time of incurred
acidosis (20). Therefore, in addition to the experiments
reported above, in which pH was changed before measurement of
0, acidosis was also induced during
submax. However, the results showed that the time
point at which pH was decreased did not alter either
Cr or max (data not shown).
Reversibility of the effects of Pi.
The reversibility of the effect of Pi on the Cr shuttle was
investigated in three rats. Measurements of respiration
(0, submax,
Cr, and max) were performed in
the presence of 20 mM Pi. Respiratory rates were remeasured
in 3 mM Pi and were nearly identical to those measured in
20 mM Pi (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The main finding in this study was that increases in [Pi] and [H+] reduce Cr-stimulated respiration in fibers from skeletal muscle. The mechanisms and the implications for muscle energetics will be discussed.
Effect of elevated [Pi]. During high rates of work, [H+] and [Pi] may increase three- and sevenfold, respectively. Theoretically, one would expect these changes to enhance oxidative phosphorylation because of increased proton-motive gradient and a decreased phosphorylation potential. Although small changes in these parameters may increase oxidative phosphorylation, previous studies (6, 24), as well as the present data, suggest that large changes may negatively affect mitochondrial function. One of the major findings in the present study was that the stimulating effect of Cr on respiration at submaximal [ADP] was reduced at elevated [Pi]. The results are in agreement with previous findings in skinned cardiac fibers (24). Therefore, it is clear that, in both skeletal and cardiac muscle, the effectiveness of the mitCK system is seriously impaired in the presence of increased concentrations of Pi, resulting in altered control of mitochondrial respiration. It seems likely that the mechanism for the reduced effect of Cr is related to a dissociation of mitCK. Previous studies have shown that, when [Pi] increases >5 mM, mitCK dissociates from the inner mitochondrial membrane (6, 25), probably because of competition for ionic binding with Pi (25). The dissociated form of mitCK has been shown to have roughly two to three times lower activity than the bound form (6, 15), and Pi-induced dissociation of mitCK may, therefore, explain the impaired function of the Cr shuttle.
During HI exercise, there is a large increase in [Pi], andCr is expected to decrease. However,
measurements of Cr in muscle samples taken at
fatigue demonstrated that Cr actually increased
(21). This is likely related to a reversal of the inhibition during the process of fiber preparation, because the fibers are exposed to low Pi during this period (~1 h).
In the present study, we examined the reversibility of the
Pi-induced decrease in Cr. Contrary to
expectations, the reduction in Cr was not reversed
when fibers were transferred to 3 mM Pi. The lack of
reversibility may be related to the short period of incubation at low
Pi (7 min).
Effect of acidosis.
In many previous studies on isolated mitochondria, acidosis has been
shown to impair the maximal rate of oxidative phosphorylation (4,
5, 7, 9). In skinned cardiac fibers, acidosis (at low
[Pi]) reduced both ADP-stimulated
(submax and max) and
non-ADP-stimulated respiration (0)
(24). In contrast to cardiac fibers, the results from this
study demonstrate that acidosis affects neither ADP-stimulated
respiration nor 0 in fibers from skeletal muscle.
Therefore, it appears that mitochondria in skeletal muscle are better
protected from the effects of acidosis than in cardiac muscle. This
seems reasonable because acidosis is a much more frequent phenomenon in
skeletal muscle than in cardiac tissue.
max when it was
induced before activation by ADP but not when acidosis was induced in the activated state (20). The results from this study on
muscle fibers demonstrate that respiration was not affected when
acidosis was evoked in activated or in quiescent fibers (i.e., before
addition of ADP). The results indicate that mitochondria are more
susceptible to acidosis when they are isolated from the tissue than
during in situ conditions (i.e., skinned fibers).
Studies on isolated cardiac mitochondria have shown conflicting results
regarding the effect of H+ on mitCK. Although one study
suggests that Pi and H+ have opposite effects
on binding of mitCK to the inner membrane (25), it has
also been shown that Pi and H+ both increase
the soluble fraction of mitCK (10). Data on skinned muscle
fibers agree with the latter finding. Acidosis was shown to reduce
Cr-stimulated respiration, both in cardiac muscle (24) and
in skeletal muscle (present study). The mechanism may be similar to
that of Pi, i.e., dissociation of mitCK from the inner
mitochondrial membrane. Another possibility is that H+
influences Cr-stimulated respiration because of its involvement in the
CK reaction (PCr + ADP + H+ 
Cr + ATP).
Increased [H+] would counteract the effect of Cr and thus
reduce local [ADP] and respiration.
The major finding in the present study was that increased
[Pi] and [H+], either in combination or
independently, reduces Cr-stimulated oxygen utilization in skeletal
muscle fibers at submaximal [ADP], but not at maximal [ADP]. This
indicates that the function of the Cr shuttle is deteriorated. The Cr
shuttle has an important role in amplifying the ADP signal in the
control of oxidative energy production. The reduced stimulation of
respiration by Cr may be overcome by increased [ADP]. However,
because ADP is an important trigger of anaerobic energy-yielding
processes and also a putative factor in fatigue (14),
increased levels of ADP may disturb cellular function.
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ACKNOWLEDGEMENTS |
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The present study was supported by grants from the Swedish National Centre for Research in Sport, Swedish Medical Research Council Grant 13020, and Estonian Science Foundation Grant 4928.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Sahlin, Dept. of Physiology and Pharmacology, Karolinska Institutet, Box 5626, SE-11486 Stockholm, Sweden.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 18, 2002;10.1152/japplphysiol.01132.2001
Received 13 November 2001; accepted in final form 12 January 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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