Department of Physiology and Pharmacology, Karolinska Institute, and Department of Sports and Health Science, Stockholm University College of Physical Education and Sports, S-11486 Stockholm, Sweden
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ABSTRACT |
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Oxidative phosphorylation of isolated rat
skeletal muscle mitochondria after exposure to lactic acidosis in
either phosphorylating or nonphosphorylating states has been evaluated.
Mitochondrial respiration and transmembrane potential
(m) were
measured with pyruvate and malate as the substrates. The addition of
lactic acid decreased the pH of the reaction medium from 7.5 to 6.4. When lactic acid was added to nonphosphorylating mitochondria, the
subsequent maximal ADP-stimulated respiration decreased by 27%
compared with that under control conditions
(P < 0.05), and the apparent
Michaelis-Menten constant
(Km) for ADP
decreased to 10 µM vs. 20 µM (P < 0.05) in controls. In contrast, maximal respiration and ADP
sensitivity were not affected when mitochondria were exposed to
acidosis during active phosphorylation in state 3. Acidosis
significantly increased mitochondrial oxygen consumption in state 4 (post-state 3), irrespective of when acidosis was induced. This effect
of acidosis was attenuated in the presence of oligomycin. The addition
of lactic acid during state 4 respiration decreased
m by 19%. The ratio between
added ADP and consumed oxygen (P/O) was close to the theoretical value
of 3 in all conditions. The addition of potassium lactate during state
3 (i.e., medium pH unchanged) had no effect on the parameters measured.
It is concluded that lactic acidosis has different effects when induced
on nonphosphorylating vs. actively phosphorylating mitochondria. On the
basis of these results, we suggest that the influence of lactic
acidosis on muscle aerobic energy production depends on the
physiological conditions at the onset of acidity.
skeletal muscle; hydrogen ions; lactate; adenosine 5'-diphosphate sensitivity; oxygen consumption
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INTRODUCTION |
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DURING REST AND EXERCISE at moderate intensity the major part of the energy demand in skeletal muscle is met by aerobic processes, but during high-intensity exercise the energy demand exceeds oxidative capacity and lactic acid formation will contribute significantly to energy production. Lactate formation is associated with a stoichiometric formation of H+, and the change in muscle pH is related to lactate accumulation and inversely related to buffer capacity. During high-intensity exercise muscle pH may decrease to 6.4 (24). Many studies have demonstrated that the development of muscle fatigue during exercise is correlated with a decrease in intracellular pH (9). It has been shown that acidosis changes the kinetics of muscle contraction (1, 39), which could have a profound effect on muscle performance.
In addition to its effect on the contraction processes, acidosis may also reduce muscle ATP production capacity. The activities of several important glycolytic enzymes are reduced during the acidotic condition (9). Aerobic ATP production also appears to be sensitive to acidosis. Data obtained from 31P-NMR spectroscopy studies on skeletal and cardiac muscles demonstrated marked metabolic deterioration [e.g., slower recovery of phosphocreatine (PCr), decreased PCr/Pi ratio, and decreased maximum oxygen consumption] during metabolic and hypercapnic acidosis (12, 21, 29), indicating impaired aerobic capacity in acidotic muscle in vivo. However, the 31P-NMR technique is not able to clarify whether the impairment of mitochondrial function is caused by acidosis per se or occurs secondarily to some other effects of acidity (e.g., decreased substrate supply). The influence of acidosis on oxidative phosphorylation was also investigated in numerous in vitro studies on isolated mitochondrial preparations exposed to different incubation medium pH conditions. This method is suitable to study the direct effects of acidosis on mitochondrial function itself. However, the results of different studies vary. In some studies maximal respiration was reduced when pH decreased to 6.3-6.5 (6, 10, 14, 16, 17), but in other studies the effect of acidosis on maximal respiration was negligible (23, 26, 27, 29, 32, 38). The reasons for these disparate findings are unclear, and the mechanisms by which acidosis affects mitochondrial function remain, as yet, incompletely understood.
A variety of methodological approaches have been used to investigate the effects of acidity on isolated mitochondria. The influences of the composition of incubation media, exposure time, and type of acid used to produce the experimental pH alterations on mitochondrial response to acidosis have been investigated (14, 17, 23). However, the influence of mitochondrial functional state (i.e., phosphorylating or nonphosphorylating mitochondria) at the point when acidotic conditions are induced has not been investigated. In all previous studies on isolated muscle mitochondria, acidosis was induced before the addition of ADP (i.e., acid was added to nonphosphorylating mitochondria in state 4). However, during sustained exercise, intracellular acidosis occurs when the mitochondrial oxygen consumption and free-ADP concentration are elevated. To mimic the physiological situation during sustained exercise, lactic acid should be added to mitochondria during active phosphorylation (i.e., in state 3, in the presence of ADP). To our knowledge this experiment has not yet been performed.
Histological studies have shown that skeletal muscle mitochondria react immediately to electrical stimulation by an increase in the size and proliferation of the cristae mitochondriales (25). These early morphological alterations may be related to conformational and spatial changes in membrane proteins, which could affect the susceptibility of mitochondrial function to acidosis. Thus we aimed in the following set of experiments to test the hypothesis that the effect of acidosis on oxidative phosphorylation is dependent on the mitochondrial functional state at the point of acidification. The purpose of this study was also to elucidate the mechanisms by which acidosis affects aerobic energy production.
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MATERIALS AND METHODS |
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Animal care and feeding. Eighteen male Sprague-Dawley rats (initially 250-300 g; BKl:SD) obtained from B & K Universal (Sollentuna, Sweden) were housed three per cage at 23°C on a cycle of 12 h of light and 12 h of darkness. Animals were given free access to a B & K Universal standard rat and mouse diet and tap water. 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 experimental protocol was approved by the local ethical committee on animal experiments.
Isolation of mitochondria.
Animals were anesthetized with pentobarbital sodium (100 mg/kg body wt
ip) and were killed between 11:00 AM and 2:00 PM. Both soleus muscles were dissected and divided into two parts. One part
(10-15 mg) was quenched in liquid nitrogen, freeze-dried, and
stored at 70°C. The specimens were later dissected from
solid nonmuscle constituents, powdered, and analyzed for muscle citrate synthase activity by a technique described previously (33). The
remaining part (115-170 mg) was used for the isolation of mitochondria by the method of Tonkonogi and Sahlin (35). Briefly, muscle specimens were disintegrated with scissors, homogenized in the
presence of bacterial proteinase (Nagarse; E.C. 3.4.21.62) and
fractionated by differential centrifugation. The final mitochondrial pellet was resuspended (0.4 µl/mg initial muscle) in a medium consisting of (in mM) 225 mannitol, 75 sucrose, 10 Tris,
and 0.1 EDTA, pH 7.40 (7.7-12.4 mg protein/ml). Before incubation,
the mitochondrial suspension was kept on ice. An aliquot of the
suspension (10 µl) was taken for measurements of protein content and
citrate synthase activity. The protein concentration in mitochondrial suspension was determined with a commercial kit (kit 690-A; Sigma Diagnostics). By using citrate synthase as a mitochondrial marker it
was possible to estimate the percentage of mitochondria freed from the
muscle and calculate mitochondrial oxygen consumption per weight of muscle.
Measurement of oxygen consumption. The respiration rates of isolated mitochondria were measured with a Clark-type electrode (DW1; Hansatech) in a water-jacketed glass chamber of 0.5-ml capacity equipped with magnetic stirring. A temperature of 25°C was maintained in the chamber. The measurements were carried out in a reaction medium containing (in mM) 225 mannitol, 75 sucrose, 10 Tris, 10 KCl, 10 K2HPO4, 0.1 EDTA, 5 pyruvate, and 2 malate, pH 7.40. Pyruvate is a substrate with a known high oxidation rate in rat skeletal muscle mitochondria. The transport of pyruvate under saturating conditions is not carrier mediated or pH dependent in the range studied here (22). The solubility of oxygen in the medium was considered to be equal to 237.5 µM.
Respiration was initiated by the addition of 7.5 µl of the mitochondrial suspension to the reaction medium, and a conventional respiratory experiment with transitions from state 4 to 3 to 4 was performed. State 3 was initiated by adding ADP (final concentration 800 µM). The respiratory control ratio (RCR) was calculated as the ratio of the respiratory rate in state 3 to the rate of oxygen uptake after exhaustion of ADP (state 4). The ratio between phosphorylated ADP added and oxygen consumed (P/O ratio) and the ADP concentration at half-maximum respiration during the deceleration phase of state 3 (Km for ADP) were determined by the method of Chance and Williams (5). The methodological variation for measurement of respiratory rates was determined from the data for different animals. The coefficients of variation, including the variation between animals, were 6.0 and 10.6% for respiration rates in states 3 and 4, respectively. Changes in the pH of the reaction medium were induced by the addition of L-lactic acid to the reaction chamber either before (2.4 ± 0.2 min) the initiation of state 3 or during maximal ADP-stimulated respiration (state 3). The final concentration of lactate in the medium was 8 mM, which is within the physiological range in exercising muscle. The effects of excess lactate ions per se on mitochondrial function were studied by adding potassium lactate (final medium concentration 8 mM) to the reaction medium during state 3 respiration. In one series of experiments L-lactic acid was added to the mitochondria respiring in state 4 in the absence (control) or presence of atractyloside (50 µM) or oligomycin (3 µg/ml). The concentrations of atractyloside and oligomycin were sufficient to completely inhibit adenine nucleotide translocase (AAT) and F1-ATPase, respectively, as indicated by a lack of increase in the respiratory rate after further addition of 800 µM ADP. After each experiment the pH of the reaction mixture was determined. The pH was 7.40 ± 0.01 and 6.38 ± 0.01 without and with the addition of L-lactic acid, respectively. The osmolarity of the reaction medium was maintained constant in all experiments by adjusting the osmolarity of additives.Estimation of
m.
The mitochondrial membrane potential
(
m) was estimated from the
distribution of cationic dye rhodamine 123 (Rh 123) between mitochondrial matrix space and the external medium. The proportion of
Rh 123 that is taken up into the matrix was determined from the
difference in absorbency at 495 and 516 nm (7) in 0.5 ml of the
above-described reaction medium containing 0.6 µM Rh 123. The
incubations were carried out at 25°C. Membrane potential was estimated assuming distribution into a matrix space of 1 µl/mg mitochondrial protein (7).
Chemicals. All chemicals were obtained from Sigma or Merck.
Data analysis. All values reported are means ± SE. Differences between means were tested for statistical significance by ANOVA with a repeated-measure design, which was followed by Fisher's post hoc tests. Significance was accepted at the 5% level.
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RESULTS |
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The mitochondrial yield, calculated from the fraction of muscle citrate
synthase activity recovered in the isolated mitochondria, was 20.2 ± 1.1%. The high degree of coupling (RCR averaged 15 in control
conditions) and a P/O ratio close to 3 (Table
1) demonstrated that the isolated
mitochondria were functionally well preserved (18). The addition of ADP
decreased the mitochondrial membrane potential by 14.7 ± 0.8%
(n = 4).
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The duration of state 3 respiration was deliberately prolonged by
adding a relatively large amount of ADP (400 nmol). By this procedure
state 3 respiration was maintained for 6-7 min and the dependence
on time could be analyzed. The average rate of respiration at the end
of state 3 (4.5 min after addition of ADP) was not significantly
different from the initial rate (1.5 min after addition of ADP), either
in controls or when L-lactic acid was added before ADP.
When L-lactic acid was added to the mitochondria during
state 3, the rate of respiration 4.5 min after the addition of ADP was 5% higher (P < 0.05) than the
initial rate. There was obviously no time-dependent
decrease in maximal respiratory rate during acidotic conditions. Figure
1 illustrates results from
experiments in which mitochondria were exposed to acidosis and excess
lactate ion during either state 3 or state 4 respiration. Maximal
respiration was reduced to 73% of the control
(P < 0.05) when lactic acid was
added to nonphosphorylating mitochondria (i.e., state 4). However, when
L-lactic acid was added during state 3, the maximal respiration rate was not changed.
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The results demonstrate that acidosis increased state 4 respiration
(post-state 3) both when L-lactic acid was added before ADP
(+92% vs. control; P < 0.05) and
when it was added after ADP (+104% vs. control; P < 0.05) (Fig. 1).
The addition of L-lactic acid to the mitochondria during
state 4 respiration decreased m by 19.2 ± 1.2%
(n = 4). The possible mechanism of the
increased state 4 respiration during acidosis has been investigated by
using atractyloside and oligomycin, which are specific inhibitors of AAT and F1-ATPase, respectively.
Oligomycin or atractyloside under control conditions did not affect
oxygen consumption in state 4. The addition of L-lactic
acid in the presence of atractyloside increased the rate of state 4 respiration to the same extent as it increased that of controls. In
contrast, the increase in oxygen consumption by acidosis was reduced by
25% in the presence of oligomycin (Fig.
2).
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After the addition of neutral potassium lactate at a concentration similar to that for L-lactic acid, with medium pH similar to that for control conditions, the respiration rates in states 3 and 4 were not significantly different from those under control conditions (Fig. 1).
The mitochondrial apparent Km for ADP under control conditions was 20 µM, which is similar to that previously observed for isolated liver mitochondria in vitro (5). The apparent Km for ADP decreased to 50% of the control (P < 0.05) when L-lactic acid was added to unstimulated mitochondria (Table 1). In contrast, when L-lactic acid was added during state 3, there was no effect on ADP sensitivity. RCR was markedly reduced during acidosis because of the increased state 4 respiration (Table 1). Neither excess lactate ion nor acidosis had a significant effect on the P/O ratio (Table 1).
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DISCUSSION |
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Findings in the present study demonstrated that mitochondrial sensitivity to lactic acidosis is dependent on the functional state of isolated mitochondria. When lactic acid was added to nonphosphorylating mitochondria, the subsequent maximal rate of oxygen consumption was reduced. However, when mitochondria were exposed to acidosis during maximal ADP-stimulated respiration, the maximal respiration rate was maintained at a level similar to that for the control condition. Previous studies on perfused skeletal and cardiac muscles have shown that oxidative capacity is reduced when the pH of the perfusion media is decreased (12, 29). With this experimental design, mitochondria are exposed to acidosis during conditions of low respiration. In contrast, when acidosis was induced by electrical stimulation or exercise (i.e., mitochondria were subjected to acidosis when cytoplasmic free-ADP concentration was elevated) the maximum rate of mitochondrial ATP production was unchanged (15, 31, 37). The results of the present study demonstrate that the functional state of mitochondria is critical for the sensitivity to acidosis and may, therefore, offer an explanation for the disparate findings in these studies on intact muscle.
Acidosis increased mitochondrial oxygen consumption in state 4 irrespective of when acidosis was induced. According to the chemiosmotic theory, state 4 respiration is due to back leakage of protons through the inner membrane of the mitochondrion and is controlled mainly by the proton permeability of the inner membrane and to some extent by dehydrogenases and translocases (11). An increased permeability of the inner mitochondrial membrane to H+ would increase oxygen consumption during state 4. Previous studies have demonstrated that leakage of protons can occur through the ATP synthetase (19, 20). The partial protective effect of oligomycin against an increase in state 4 at lowered pH indicates that part of the increased state 4 oxygen consumption is caused by proton leakage through the ATP synthetase. Because state 4 respiration was not affected by the addition of oligomycin under control conditions, the effect of acidosis on state 4 respiration may, partially, be attributed to the induction of ATP synthetase permeability to protons.
It is well documented that native and reconstituted ADP/ATP translocase, under certain conditions (e.g., in the presence of free fatty acids or Ca2+), can participate in proton transport through the membranes by functioning as a H+-specific conductor (3, 4, 13). In these studies, it was shown that inhibitors of AAT suppressed AAT-mediated proton transport (3, 4, 13). However, we found that lactic acidosis induced in the presence of atractyloside, a potent inhibitor of AAT, increased the rate of oxygen consumption to the same extent as it increased that in controls. This finding suggests that the augmented mitochondrial proton leak during acidotic conditions occurs through mitochondrial components or structures other than AAT.
Several studies have demonstrated an overshoot in PCr during the recovery phase after high-intensity exercise (8, 28, 30). An overshoot in PCr may indicate that cytoplasmic free-ADP concentration is lower than it is at rest. Because cytoplasmic free-ADP concentration is one of the most important regulators of mitochondrial function, the mitochondrial oxygen consumption during recovery after high-intensity exercise should be lower than it is at rest. However, after termination of exercise, whole body oxygen uptake is maintained above the basal level. It is possible that the increased mitochondrial proton leakage during acidosis may contribute to this phenomenon.
The futile cycle of proton pumping and leakage across the mitochondrial
membrane requires oxygen utilization, which is unrelated to ATP
synthesis. One may therefore expect that the P/O ratio should be
reduced during acidosis. However, the P/O ratios during acidosis and
control conditions were similar. This phenomenon can be explained by a
rapid switch from futile proton leakage to proton flux-mediated ATP
production during the state 4-to-3 transition. The addition of ADP will
decrease the protonmotive force, as indicated by the observed drop in
m. As a consequence, the
rate of proton leakage will decrease during state 3 (2), which explains
why the P/O ratio is maintained unchanged during acidosis.
Our finding that acidosis does not affect the P/O ratio in isolated
skeletal muscle mitochondria is in agreement with some previous studies
on isolated mitochondria (23, 32) and with recent observations on
perfused cat soleus muscle by the NMR technique (12). However, some
studies of isolated mitochondria have found a lower P/O ratio during
acidosis (14, 17). The reason for this disparity is not clear. The
modulation of chemical efficiency in the energy transfer by ADP may
provide an advantage in that the rate of oxidative ATP production can
increase without great changes in oxygen consumption rate when energy
demand increases. This will reduce the fluctuations in cytosolic
free-ADP concentration and, as a consequence, in
G (change in Gibbs free
energy) of the ATP hydrolysis.
An interesting finding in this study was that the sensitivity of mitochondrial respiration to ADP was increased by acidosis when induced during unstimulated respiration. To our knowledge this phenomenon has not been observed previously in isolated mitochondria. However, data from NMR studies imply that the ADP control of oxidative phosphorylation is altered by acidosis. Despite a decrease in calculated free-ADP concentration during acidosis, the oxygen consumption rate and PCr concentration in noncontracting cat skeletal muscle were found to be unchanged in previous studies (1, 12). Furthermore, during stimulation of rat and cat skeletal muscle the relationship between cytosolic free-ADP concentration and estimated oxygen consumption was altered by acidosis in a manner that indicates the augmented sensitivity of oxidative phosphorylation to ADP (12, 21). In a previous study we found that mitochondrial ADP sensitivity in human saponin-skinned muscle fibers increased significantly after exhaustive high-intensity intermittent exercise with severe acidosis (36). In contrast, after prolonged exhaustive exercise without lactic acidosis, the sensitivity of mitochondrial respiration to ADP was unchanged (34). Taken together these findings support the idea that intracellular acidosis, under some conditions, can increase mitochondrial ADP sensitivity.
In summary, the present study demonstrated that lactic acid has different effects when added to nonphosphorylating or actively phosphorylating mitochondria. Nonphosphorylating mitochondria appeared to be more sensitive to acidosis. The maximal rate of respiration was reduced, and sensitivity to ADP was increased, when nonphosphorylating mitochondria were exposed to acidosis. Respiration in state 4 increased during acidotic conditions irrespective of the mode of lactic acid addition.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Anita Matthias and Dr. Jan Nedergaard for
their help with the technique to measure
m.
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FOOTNOTES |
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The present study was supported by grants from the Swedish National Center for Research in Sport, the Swedish Medical Research Council, and the Karolinska Institute.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. Sahlin, Dept. of Physiology and Pharmacology, Karolinska Institute, Box 5626, S-11486 Stockholm, Sweden (E-mail: kent.sahlin{at}fyfa.ki.se).
Received 29 December 1998; accepted in final form 30 April 1999.
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