Vol. 24, No. 4, pp. 657-661, 1992 Printed in Great Britain. All rights reserved
0020-711X/92$5.00+ 0.00 Copyright 0 1992Pergamon Press plc
Int. J. Biochem.
REGULATING EFFECT OF MITOCHONDRIAL LACTATE DEHYDROGENASE ON OXIDATION OF CYTOPLASMIC NADH VIA AN “EXTERNAL” PATHWAY IN SKELETAL MUSCLE MITOCHONDRIA ANNA SZCZFSNA-KACZMAREK Department of Physiology, “Jedrzej Sniadecki” Academy of Physical Education, Wiejska 1, 80-336 Gdatisk-Oliwa, Poland (Received 17 May 1991) Abstract-l. The specific activity of lactate dehydrogenase of skeletal muscle mitochondria was found to be 2.5 times lower than specific activity of total NADH-cytochrome c reductase. 2. The specific activity of mitochondrial LDH in skeletal muscle mitochondria was almost equal to the activity of rotenone-insensitive NADH-cytochrome c reductase. 3. Mitochondrial LDH acting as an oxidase of lactate to pyruvate may feed an “external” pathway, but the activity of the mitochondrial enzyme is a limiting factor in oxidation of lactate-derived NADH. 4. Mitochondrial LDH acting as a reductase of pyruvate to lactate successfully competes with an “external” pathway for cytoplasmic NADH. 5. Exogenous NADH oxidation via an “external” pathway was inhibited by pyruvic acid. This inhibition was overcome by addition of oxamic acid or hydrazine.
INTRODUCTION
Observations of production of lactate in muscle during exercise in aerobic conditions have been documented (Connett et al., 1984; Brooks, 1986; Wolfe et al., 1987). However, experimental evidence reveals that the decrease in NAD: NADH (an indicator of tissue hypoxia) and in ATP : ADP ratio is prominent in the cell at ~0, below 5 mmHg (De Grott and Noll, 1987; Chance et al., 1985). Furthermore, there is evidence, that in the muscle p0, during intense contractions appears to be above the minimal critical level of 8-10 mmHg, so pOZ in the working muscle is adequate (Stainsby, 1986). On the basis of these observations, the general question is why muscles produce lactic acid during contraction while there is adequate O2 in mitochondria. We expected an answer to this question by studying reactions involved in reoxidation of cytoplasmic reducing equivalents contributing to the availability of NAD in the cell cytoplasm. The role to removal of NADH formed in the cytosol may represent one of the main factors regulating the rate of aerobic production of lactate in the glycolyzing cells. Previously we provided direct evidence that in skeletal muscle, cytosolic hydrogen atoms can reach oxygen by a pathway which involves the action of rotenone insensitive NADH-cytochrome b, reductase from the outer mitochondrial membrane, cytochrome c from intermembrane space, cytochrome c and cytochrome oxidase from inner mitochondrial membrane (the “external” pathway) (Szczpsna-Kaczmarek et al., 1984). It is known from our experiments that cytosolit lactate dehydrogenase was able to feed the “external” pathway with NADH. The activity of oxidation
of lactate-derived
hydrogens via an “external” pathway was not inhibited by acidification nor by ammonia (Szczesna-Kaczmarek, 1989). In a recent study we pointed out the importance of mitochondrial lactate dehydrogenase in the operation of the hydrogen transporting shuttle, when it was involved in direct oxidation of lactate by skeletal muscle mitochondria (Szczesna-Kaczmarek, 1990). Taking into consideration the knowledge that the reoxidation of cytosolic NADH by the respiratory chain mainly proceeds via the malate-aspartate cycle, the mutual relations between mitochondrial reactions of malate-aspartate shuttle and reaction of mitochondrial lactate dehydrogenase were investigated (Szczesna-Kaczmarek and Szydlowski, 1990). From our results it is known that the mitochondrial LDH shuttle may act simultaneously with the malate-aspartate shuttle and the activity of both shuttles was approximately additive in muscle mitochondria, but in liver mitochondria we did not find a summation activity of oxidation substrates composing both transporting shuttles. Some of problems of general question have been explained in previous experiments but as others still remain unsolved, we determined to try to find an explanation in this study. One of the questions is whether mitochondrial lactate dehydrogenase may feed an “external” pathway. Closely related to this question is whether the lactate-derived hydrogens oxidation via rotenone insensitive NADH-cytochrome c reductase is limited by mitochondrial LDH activity. The third problem is the mutual relation between oxidation of extramitochondrial NADH oxidation via an “external” pathway and via mitochondrial LDH. 657
ANNASZCZ~SNA-KACZMAREK
658 Table 1. Specific activity
of the membrane
Activity of lactate dehydrogenase in nmol NADH x mg prot.-’ x min-’ Forward reaction ovruvate-lactate .I
Activity of NADH-cytcchrome e reductase in nmol cytochrome c reduced x mg prot.-’ x min-’
Backward reaction lactate+avruvate
Total
19.8 f 4.5
178.1 k 21.4
.~
69.6 f 19.1 The experimental experiments.
enzymes in rat skeletal muscle mitochondria
conditions
MATERIALS AND
are given
in Materials
and Methods.
Retenoneinsensitive
Rotenonesensitive
55.1 57.1
123 f 14.2
The values
are given as the means f SE, n = 6
The chemicals used were of the highest purity and were obtained from Sigma Chemicals Co, St Louis, MO., U.S.A., or Boehringer Mannheim GmbH.
buffer, 2 mM EDTA, 0.1% BSA, l-2 mg mitochondrial protein. Respiratory control index (RCI) and ADP:O ratio were calculated from oxygen electrode traces according to Estabrook (1967).
Preparation
Assay of NADH-cytochrome
METHDOS
of mitochondria
Mitochondria were isolated in the cold from the rat skeletal muscle of the hind legs. The tissues were immediately placed into cold 150 mM KC1 and dissected free of connective tissues and fat and were weighed. They were chopped and homogenized in medium pH 7.4 containing: 210 mM mannitol, 70 mM sucrose, 50 mM Trizma-HCI, pH 7.4, 10 mM EDTA, 0.2% BSA and 50 U of heparin in 1 ml medium. Homogenization was performed by hand using a Teflon-glass homogenizer. The resulting homogenate (1: 10) was centrifuged for 10 min at 600 g to remove nuclei and cell debris. After repeating this centrifugation the mitochondria were isolated from the resulting supernatant by centrifugation for 10 min at 12,OOOg.The mitochondrial pellet was rinsed with medium containing: 210 mM mannitol, 70 mM Trirma-HCl, pH 7.4, and centrifuged again for 10min at 12,OOOg.The washing of mitochondria was repeated three times. This procedure yielded well coupled mitochondria with ADP:O ratio of 2.7 with pyruvate plus malate as substrates. The protein content in mitochondrial suspension was determined by the method of Lowry et al. (1951) with bovine serum albumin (BSA) as a standard. Assay of lactate dehydrogenase
activity
LDH activity in mitochondria was measured spectrophotometrically in both directions: following the oxidation of L-lactate to pyruvate and reduction of NAD to NADH (backward reaction) in the medim containing: Trizma-HCl buffer, pH 8.2, 1OOmM with 0.2% Triton-X-100, and forward reaction-following reduction of pyruvate to lactate in the medium containing: 50 mM phosphate buffer, pH 7.4, with 0.2% Triton-X-100. The changes in absorbance at 340nm were determined using Pye-Unicam SP-8-100 u.v./vis. spectrophotometer. Assay of respiratory
activity of skeletal muscle mitochondria
Oxidation of different respiratory substrates was measured by monitoring of oxygen consumption with a Clark oxygen electrode (Gilson Polarograph, U.S.A.). The reaction medium contained in 2ml volumes: 50mM Trirma-HCl buffer, pH 7.0, 40 mM KCl, 5 mM phosphate
Table 2. Comparison
of activity
oxidations
RESULTS
The data in Table 1 show that the mitochondrial lactate dehydrogenase in skeletal muscle mitochondria when compared with the total activity of NADH-cytochrome c reductase has 2.5 times and 8.9 times lower specific activity assayed as a reductase and oxidase respectively. Similarly the specific activity of rotenone insensitive and sensitive NADHcytochrome c reductase is 2.5 times and 6 times higher respectively than specific activity of mitochondrial LDH (measured as an oxidase). These data confirm the finding of the high activity of external NADH oxidation in the presence of cytochrome c by skeletal muscle mitochondria (Fig. 1) and explains the reason of the low activity of lactate-derived NADH oxidation via an “external” pathway (Table 3). From the results above it is also clear that the activity of mitochondrial LDH is a limiting factor in oxidation of LA-derived hydrogens via an “external” pathway. For measuring the maximum activity of oxidation of LA-derived reducing equivalents in this manner, the cytoplasmic fraction containing LDH was added to the experimental medium (Table 3). The obtained result shows that the cytoplasmic treatment is not sufficient to achieve the maximum rate of LA-derived hydrogens oxidation (Table 3). These phenomena suggest a possible integration of mitochondrial LDH (but not externally added LDH) with the function of mitochondrial enzyme system.
of L-lactate plus NAD with pyruvate
State metabolic 3 rate n atoms 0, x ma prot-’ x min-’
Substrates Pyruvate plus malate L-Lactate plus NAD
5mM 1OmM
The experimental
conditions
c reductase activity
NADH-cytochrome c reductase activity was measured by determining the reduction of cytochrome c at 550 nm. The medium contained 100 mM phosphate buffer, pH 7.4, 1 mM KCl, 0.05 mM cytochrome c and 0.1 mM NADH. Rotenone sensivity was tested using 0.002 mM rotenone. Rotenone-sensitive NADH-cytochrome c reductase activity was calculated as the difference between the total and rotenone-insensitive activity (Sottocasa et al., 1967).
Respiratory
plus malate in skeletal muscle control
(WI)
index
ADP:O ratio
5mM
1mM
74.7 * 12
56 f 17.4 are given in Materials
and Methods.
3.9 + 0.6
2.5 f 0.17
1.9 f 0.25
1.64 & 0.27
The values are means f SE, n = 6 experiments.
muscle mitochondrial LDH
Skeletal
659
NADH 150 nM
NADH 150 nM
Pyruvate
500 pM
NADH 150 nM NADH 150 nM
NADH 150 nM
Oxamic acid 2 mM
1 Pyruvate 500 pM NADH 150 nM
(D)
NADH 150 nM
L 26
T 180flat
I
180s
Fig. 1. Experiments A-D: rotenone-insensitive respiration in rat skeletal muscle mitochondria.NADH used as a respiratory substrate. 2.7 mg mitochondrial protein incubated in the medium containing: 50 mM Trirma-HCI pH 7.0, 40 mM KCl, 5 mM phosphate buffer, 2 mM EDTA, 0.1% BSA, 4 PM cytochrome c, 2.5 FM rotenone in 2 ml medium. Other additions as indicated in the figures. Temp 23°C. Oxygen uptake is expressed as n atom 0, x mg prot.-’ x min-‘.
A very interesting situation occurred when externally added NADH was oxidized by an “external” pathway in the presence of pyruvate (Fig. 1). This oxidation appeared to be sensitive to pyruvate. The observed inhibition can be attributed to the lack of NADH for the “external” oxidation pathway due to reduction of pyruvate to lactate catalyzed by mitochondrial lactate dehydrogenase. The inhibition was overcome by addition of oxamic acid-an
inhibitor of LDH activity, or hydrazine-a reagent binding pyruvic acid. Mitochondrial lactate dehydrogenase in this oxidation pathway acts as a reductase and, as shown in this work, its specific activity is almost equal to the specific activity of rotenone-insensitive NADH-cytochrome c reductase (Table l), due to mitochondrial LDH successfully competing with an “external” pathway for cytoplasmic NADH.
ANNA SZCZFSNA-KACZMAREK
660 Table 3. Comparison
of activity
oxidations
of extramitochondrial “external” pathway
NADH
with LA-derived
Oxidation rate in n atoms 0, x mg prot.-’ x min-’
Substrates NADH extramitochondrial NADH; LA-derived (in reaction catalyzed
I mM
by mitochondrial
LDH)
NADH; LA-derived (in reaction catalyzed by mitochondrial LDH supplemented with cvtoplasmic LDH)”
NADH
via an
% of Control
29.9 i 2.3
100
7.5 i 2.9
25
16.0 + 3.2
53
Oxidation of NADH was measured by monitoring of oxygen uptake with a Clark oxygen electrode (Gilson Polarograph). The reactions medium contained in 2 ml volumes: 50 mM T&ma-HCI, pH 7.0, 40 mM KCI, 5 mM phosphate buffer, 2 mM EDTA, 0.1% BSA, 4 PM cytochrome c, 2.5 PM rotenone, 2.7 mg mitochondrial protein. “Cytoplasmic, I .5 mg protein.
DISCUSSION It is known that production of lactic acid and output in skeletal muscle during repetitive contractions due to the fact that activation of glycolysis is more rapid than activation of oxidative phosphorylation. In these conditions p02 in muscle is adequate (Stainsby, 1986; Pirnay et al., 1973; Connett et al., 1984). This results in a transient elevation of NADH in the cytoplasm. The muscle cytosolic NAD content is, however, only of the order of 1/5Crl/lOO of that needed per min of intense contractions of muscle (Sahlin, 1983). Thus, rapid re-oxidation of NADH is necessary in order for glycolysis to proceed (Borst, 1963; Schantz and Henrikson, 1987). As previously indicated, and as shown in Table 2, mitochondrial lactate dehydrogenase in muscle mitochondria is integrated with the function of mitochondrial inner membrane-bound NADH oxidase system and is involved in direct oxidation of L-lactate, simultaneously the reducing equivalents represented in lactic acid are directly transported from cytoplasm to mitochondrial inner membrane. Thus, the mitochondrial LDH plays the role of an additional system transporting hydrogens from cytoplasm to mitochondria. The data in this work indicate the successive important role of mitochondrial LDH in skeletal muscle cell. On the basis of the present data we suggest a controlling role of mitochondrial LDH regarding extramitochondrial oxidation of NADH in the presence of cytochrome c, via rotenone-insensitive NADH-cytochrome c reductase. Extramitochondrial oxidation of NADH via an “external” pathway has already been described (Sottocasa et al., 1967; Bernardi and Azzone, 1981; Bernardi and Azzone, 1982; Szczesna-Kaczmarek et al., 1984), but with regard to energy conservation (only 1 side of ATP synthesis, Bernardi and Azzone, 1982), the role of this pathway is still unclear. We suggested previously that the skeletal cell utilizes this uneconomical system only exclusively as a defence mechanism against the effect of prolonged acidosis caused by accumulation of lactic acid (Szczesna-Kaczmarek et al., 1984). In the conditions mentioned above such a form of respiration acts as a hydrogen transporting shuttle, At present we have shown the mechanism controlling the activity of the “external” pathway acting as a shuttle transporting reducing equivalents. This report shows that pyruvate exerts an inhibitory effect on external
NADH oxidation in the presence of cytochrome c due to the presence of mitochondrial LDH. The reduction rate of pyruvate to lactate was sufficient to keep extramitochondrial NADH and hinder oxidation through the “external” pathway. It may be concluded that mitochondrial lactate dehydrogenase successfully competes with an “external” pathway for cytoplasmic NAD until the lactate/pyruvate ratio reaches a value allowing extramitochondrial NADH to act as a substrate for rotenone-insensitive NADHcytochrome c reductase. REFERENCES
Bemardi P. and Azzone G. F. (1981) Cytochrome
c as an electron shuttle between the outer and inner mitochondrial membranes. J. biol. Chem. 256,7187-7192. Bemardi P. and Azzone G. F. (1982) ATP synthesis during exogenous NADH oxidation. A reappraisal. Biochim. biophys. Acia 679, 19-27.
Borst P. (1963) Hydrogen transport and transport metabolites. In Functionelle und Morphologische Organization der Zelle (Edited by Karlson P.), pp. 137-158. Springer, Berlin. Brooks G. A. (1986) Lactate production under fully aerobic conditions: the lactate shuttle during rest and exercise. Fed. Proc. 45, 2924-2929.
Chance B., Leigh J. S. Jr, Clark B. J., Maris J., Kent J., Nioka S. and Smith D. (1985) Control of oxidative metabolism and oxygen delivery in human skeletal muscle: a steady-state analysis of the work/energy cost transfer function. Proc. natn. Acad. Sci. U.S.A. 82, 83848388. Connett R. J., Gatyeski E. J. and Honig C. R. (1984) Lactate accumulation in fully aerobic, working, dog gracilis muscle. Am. J. Physiol. 246, H12&H128. De Grott H. and No11 I. (1987) Oxygen gradients: the oroblem of hvnoxia. Biochem. Sot. Trans. 15, 363-365. Esiabrook R. w. (1967) Mitochondrial respiratory control and the polarographic measurement of ADP: 0 ratio. In Methods in Enzymology (Edited by Estabrook R. W. and Pulmann H. E.) Vol. 10, pp. 4147. Academic Press, New York. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Pirnay F., Marechal R., Dujardin R., Lamy M., Deroanne R. and Petit J. M. (1973) Exercise during hypoxia and hyperbaric oxygenation. Int. Z. angew. Physiol. einschl. Arbeitphysiol. 31, 259-268.
Sahlin K. (1983) NADH and NADPH in human skeletal muscle at rest and during ischaemia. C/in. Physiol. 3, 477-485.
Schantz P. G. and Henrikson J. (1987) Enzyme levels of the NADH shuttle systems: measurements in isolated
Skeletal muscle mitochondrial muscle fiber from humans differing physical activity. Actu physiol. stand. 129, SOS-515
Sottocasa G. L., Kulenstiema B. D., Emster L. and Bergstrand A. (1967) An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol. 32, 415438. Stainsby W. N. (1986) Biochemical and physiological bases for lactate production. Med. Sci. Sports Exere. 18, 341-343. Szcqsna-Kaczmarek A., Litwitiska D. and Popinigis J. (1984) Oxidation of NADH via an “external” pathway in skeletal muscle mitochondria and its possible role in the repayment of lactacid oxygen debt. Int. J. Biochem. 16, 1231-123s.
LDH
661
Sacqsna-Kaczmarek A. (1989) Contribution of lactate dehydrogenase in hydrogen transporting shuttle under conditions simulating postexercise recovery phase. Biol. Sporr 6, 129-138. Sacqsna-Kaczmarek A. (1990) L-Lactate oxidation by skeletal muscle mitochondria. Inf. J. Biochem. 22, 617620.
Sacqsna-Kacxmarek A. and Szydlowski J. (1990) Interrelationship between mitochondrial reactions of malatcaspartate shuttle and mitochondrial lactate dehydrogenase. Biol. Sport 7, 78-92. Wolfe B. R., Graham T. E. and Barclay J. K. (1987) Hyperoxia, mitochondrial redox state, and lactate metabolism of in siru canine muscle. Am. J. Physiol. W,
(32634268.
0020-711X/92$5.00+ 0.00 Copyright 0 1992Pergamon Press plc
Int. J. Biochem.
REGULATING EFFECT OF MITOCHONDRIAL LACTATE DEHYDROGENASE ON OXIDATION OF CYTOPLASMIC NADH VIA AN “EXTERNAL” PATHWAY IN SKELETAL MUSCLE MITOCHONDRIA ANNA SZCZFSNA-KACZMAREK Department of Physiology, “Jedrzej Sniadecki” Academy of Physical Education, Wiejska 1, 80-336 Gdatisk-Oliwa, Poland (Received 17 May 1991) Abstract-l. The specific activity of lactate dehydrogenase of skeletal muscle mitochondria was found to be 2.5 times lower than specific activity of total NADH-cytochrome c reductase. 2. The specific activity of mitochondrial LDH in skeletal muscle mitochondria was almost equal to the activity of rotenone-insensitive NADH-cytochrome c reductase. 3. Mitochondrial LDH acting as an oxidase of lactate to pyruvate may feed an “external” pathway, but the activity of the mitochondrial enzyme is a limiting factor in oxidation of lactate-derived NADH. 4. Mitochondrial LDH acting as a reductase of pyruvate to lactate successfully competes with an “external” pathway for cytoplasmic NADH. 5. Exogenous NADH oxidation via an “external” pathway was inhibited by pyruvic acid. This inhibition was overcome by addition of oxamic acid or hydrazine.
INTRODUCTION
Observations of production of lactate in muscle during exercise in aerobic conditions have been documented (Connett et al., 1984; Brooks, 1986; Wolfe et al., 1987). However, experimental evidence reveals that the decrease in NAD: NADH (an indicator of tissue hypoxia) and in ATP : ADP ratio is prominent in the cell at ~0, below 5 mmHg (De Grott and Noll, 1987; Chance et al., 1985). Furthermore, there is evidence, that in the muscle p0, during intense contractions appears to be above the minimal critical level of 8-10 mmHg, so pOZ in the working muscle is adequate (Stainsby, 1986). On the basis of these observations, the general question is why muscles produce lactic acid during contraction while there is adequate O2 in mitochondria. We expected an answer to this question by studying reactions involved in reoxidation of cytoplasmic reducing equivalents contributing to the availability of NAD in the cell cytoplasm. The role to removal of NADH formed in the cytosol may represent one of the main factors regulating the rate of aerobic production of lactate in the glycolyzing cells. Previously we provided direct evidence that in skeletal muscle, cytosolic hydrogen atoms can reach oxygen by a pathway which involves the action of rotenone insensitive NADH-cytochrome b, reductase from the outer mitochondrial membrane, cytochrome c from intermembrane space, cytochrome c and cytochrome oxidase from inner mitochondrial membrane (the “external” pathway) (Szczpsna-Kaczmarek et al., 1984). It is known from our experiments that cytosolit lactate dehydrogenase was able to feed the “external” pathway with NADH. The activity of oxidation
of lactate-derived
hydrogens via an “external” pathway was not inhibited by acidification nor by ammonia (Szczesna-Kaczmarek, 1989). In a recent study we pointed out the importance of mitochondrial lactate dehydrogenase in the operation of the hydrogen transporting shuttle, when it was involved in direct oxidation of lactate by skeletal muscle mitochondria (Szczesna-Kaczmarek, 1990). Taking into consideration the knowledge that the reoxidation of cytosolic NADH by the respiratory chain mainly proceeds via the malate-aspartate cycle, the mutual relations between mitochondrial reactions of malate-aspartate shuttle and reaction of mitochondrial lactate dehydrogenase were investigated (Szczesna-Kaczmarek and Szydlowski, 1990). From our results it is known that the mitochondrial LDH shuttle may act simultaneously with the malate-aspartate shuttle and the activity of both shuttles was approximately additive in muscle mitochondria, but in liver mitochondria we did not find a summation activity of oxidation substrates composing both transporting shuttles. Some of problems of general question have been explained in previous experiments but as others still remain unsolved, we determined to try to find an explanation in this study. One of the questions is whether mitochondrial lactate dehydrogenase may feed an “external” pathway. Closely related to this question is whether the lactate-derived hydrogens oxidation via rotenone insensitive NADH-cytochrome c reductase is limited by mitochondrial LDH activity. The third problem is the mutual relation between oxidation of extramitochondrial NADH oxidation via an “external” pathway and via mitochondrial LDH. 657
ANNASZCZ~SNA-KACZMAREK
658 Table 1. Specific activity
of the membrane
Activity of lactate dehydrogenase in nmol NADH x mg prot.-’ x min-’ Forward reaction ovruvate-lactate .I
Activity of NADH-cytcchrome e reductase in nmol cytochrome c reduced x mg prot.-’ x min-’
Backward reaction lactate+avruvate
Total
19.8 f 4.5
178.1 k 21.4
.~
69.6 f 19.1 The experimental experiments.
enzymes in rat skeletal muscle mitochondria
conditions
MATERIALS AND
are given
in Materials
and Methods.
Retenoneinsensitive
Rotenonesensitive
55.1 57.1
123 f 14.2
The values
are given as the means f SE, n = 6
The chemicals used were of the highest purity and were obtained from Sigma Chemicals Co, St Louis, MO., U.S.A., or Boehringer Mannheim GmbH.
buffer, 2 mM EDTA, 0.1% BSA, l-2 mg mitochondrial protein. Respiratory control index (RCI) and ADP:O ratio were calculated from oxygen electrode traces according to Estabrook (1967).
Preparation
Assay of NADH-cytochrome
METHDOS
of mitochondria
Mitochondria were isolated in the cold from the rat skeletal muscle of the hind legs. The tissues were immediately placed into cold 150 mM KC1 and dissected free of connective tissues and fat and were weighed. They were chopped and homogenized in medium pH 7.4 containing: 210 mM mannitol, 70 mM sucrose, 50 mM Trizma-HCI, pH 7.4, 10 mM EDTA, 0.2% BSA and 50 U of heparin in 1 ml medium. Homogenization was performed by hand using a Teflon-glass homogenizer. The resulting homogenate (1: 10) was centrifuged for 10 min at 600 g to remove nuclei and cell debris. After repeating this centrifugation the mitochondria were isolated from the resulting supernatant by centrifugation for 10 min at 12,OOOg.The mitochondrial pellet was rinsed with medium containing: 210 mM mannitol, 70 mM Trirma-HCl, pH 7.4, and centrifuged again for 10min at 12,OOOg.The washing of mitochondria was repeated three times. This procedure yielded well coupled mitochondria with ADP:O ratio of 2.7 with pyruvate plus malate as substrates. The protein content in mitochondrial suspension was determined by the method of Lowry et al. (1951) with bovine serum albumin (BSA) as a standard. Assay of lactate dehydrogenase
activity
LDH activity in mitochondria was measured spectrophotometrically in both directions: following the oxidation of L-lactate to pyruvate and reduction of NAD to NADH (backward reaction) in the medim containing: Trizma-HCl buffer, pH 8.2, 1OOmM with 0.2% Triton-X-100, and forward reaction-following reduction of pyruvate to lactate in the medium containing: 50 mM phosphate buffer, pH 7.4, with 0.2% Triton-X-100. The changes in absorbance at 340nm were determined using Pye-Unicam SP-8-100 u.v./vis. spectrophotometer. Assay of respiratory
activity of skeletal muscle mitochondria
Oxidation of different respiratory substrates was measured by monitoring of oxygen consumption with a Clark oxygen electrode (Gilson Polarograph, U.S.A.). The reaction medium contained in 2ml volumes: 50mM Trirma-HCl buffer, pH 7.0, 40 mM KCl, 5 mM phosphate
Table 2. Comparison
of activity
oxidations
RESULTS
The data in Table 1 show that the mitochondrial lactate dehydrogenase in skeletal muscle mitochondria when compared with the total activity of NADH-cytochrome c reductase has 2.5 times and 8.9 times lower specific activity assayed as a reductase and oxidase respectively. Similarly the specific activity of rotenone insensitive and sensitive NADHcytochrome c reductase is 2.5 times and 6 times higher respectively than specific activity of mitochondrial LDH (measured as an oxidase). These data confirm the finding of the high activity of external NADH oxidation in the presence of cytochrome c by skeletal muscle mitochondria (Fig. 1) and explains the reason of the low activity of lactate-derived NADH oxidation via an “external” pathway (Table 3). From the results above it is also clear that the activity of mitochondrial LDH is a limiting factor in oxidation of LA-derived hydrogens via an “external” pathway. For measuring the maximum activity of oxidation of LA-derived reducing equivalents in this manner, the cytoplasmic fraction containing LDH was added to the experimental medium (Table 3). The obtained result shows that the cytoplasmic treatment is not sufficient to achieve the maximum rate of LA-derived hydrogens oxidation (Table 3). These phenomena suggest a possible integration of mitochondrial LDH (but not externally added LDH) with the function of mitochondrial enzyme system.
of L-lactate plus NAD with pyruvate
State metabolic 3 rate n atoms 0, x ma prot-’ x min-’
Substrates Pyruvate plus malate L-Lactate plus NAD
5mM 1OmM
The experimental
conditions
c reductase activity
NADH-cytochrome c reductase activity was measured by determining the reduction of cytochrome c at 550 nm. The medium contained 100 mM phosphate buffer, pH 7.4, 1 mM KCl, 0.05 mM cytochrome c and 0.1 mM NADH. Rotenone sensivity was tested using 0.002 mM rotenone. Rotenone-sensitive NADH-cytochrome c reductase activity was calculated as the difference between the total and rotenone-insensitive activity (Sottocasa et al., 1967).
Respiratory
plus malate in skeletal muscle control
(WI)
index
ADP:O ratio
5mM
1mM
74.7 * 12
56 f 17.4 are given in Materials
and Methods.
3.9 + 0.6
2.5 f 0.17
1.9 f 0.25
1.64 & 0.27
The values are means f SE, n = 6 experiments.
muscle mitochondrial LDH
Skeletal
659
NADH 150 nM
NADH 150 nM
Pyruvate
500 pM
NADH 150 nM NADH 150 nM
NADH 150 nM
Oxamic acid 2 mM
1 Pyruvate 500 pM NADH 150 nM
(D)
NADH 150 nM
L 26
T 180flat
I
180s
Fig. 1. Experiments A-D: rotenone-insensitive respiration in rat skeletal muscle mitochondria.NADH used as a respiratory substrate. 2.7 mg mitochondrial protein incubated in the medium containing: 50 mM Trirma-HCI pH 7.0, 40 mM KCl, 5 mM phosphate buffer, 2 mM EDTA, 0.1% BSA, 4 PM cytochrome c, 2.5 FM rotenone in 2 ml medium. Other additions as indicated in the figures. Temp 23°C. Oxygen uptake is expressed as n atom 0, x mg prot.-’ x min-‘.
A very interesting situation occurred when externally added NADH was oxidized by an “external” pathway in the presence of pyruvate (Fig. 1). This oxidation appeared to be sensitive to pyruvate. The observed inhibition can be attributed to the lack of NADH for the “external” oxidation pathway due to reduction of pyruvate to lactate catalyzed by mitochondrial lactate dehydrogenase. The inhibition was overcome by addition of oxamic acid-an
inhibitor of LDH activity, or hydrazine-a reagent binding pyruvic acid. Mitochondrial lactate dehydrogenase in this oxidation pathway acts as a reductase and, as shown in this work, its specific activity is almost equal to the specific activity of rotenone-insensitive NADH-cytochrome c reductase (Table l), due to mitochondrial LDH successfully competing with an “external” pathway for cytoplasmic NADH.
ANNA SZCZFSNA-KACZMAREK
660 Table 3. Comparison
of activity
oxidations
of extramitochondrial “external” pathway
NADH
with LA-derived
Oxidation rate in n atoms 0, x mg prot.-’ x min-’
Substrates NADH extramitochondrial NADH; LA-derived (in reaction catalyzed
I mM
by mitochondrial
LDH)
NADH; LA-derived (in reaction catalyzed by mitochondrial LDH supplemented with cvtoplasmic LDH)”
NADH
via an
% of Control
29.9 i 2.3
100
7.5 i 2.9
25
16.0 + 3.2
53
Oxidation of NADH was measured by monitoring of oxygen uptake with a Clark oxygen electrode (Gilson Polarograph). The reactions medium contained in 2 ml volumes: 50 mM T&ma-HCI, pH 7.0, 40 mM KCI, 5 mM phosphate buffer, 2 mM EDTA, 0.1% BSA, 4 PM cytochrome c, 2.5 PM rotenone, 2.7 mg mitochondrial protein. “Cytoplasmic, I .5 mg protein.
DISCUSSION It is known that production of lactic acid and output in skeletal muscle during repetitive contractions due to the fact that activation of glycolysis is more rapid than activation of oxidative phosphorylation. In these conditions p02 in muscle is adequate (Stainsby, 1986; Pirnay et al., 1973; Connett et al., 1984). This results in a transient elevation of NADH in the cytoplasm. The muscle cytosolic NAD content is, however, only of the order of 1/5Crl/lOO of that needed per min of intense contractions of muscle (Sahlin, 1983). Thus, rapid re-oxidation of NADH is necessary in order for glycolysis to proceed (Borst, 1963; Schantz and Henrikson, 1987). As previously indicated, and as shown in Table 2, mitochondrial lactate dehydrogenase in muscle mitochondria is integrated with the function of mitochondrial inner membrane-bound NADH oxidase system and is involved in direct oxidation of L-lactate, simultaneously the reducing equivalents represented in lactic acid are directly transported from cytoplasm to mitochondrial inner membrane. Thus, the mitochondrial LDH plays the role of an additional system transporting hydrogens from cytoplasm to mitochondria. The data in this work indicate the successive important role of mitochondrial LDH in skeletal muscle cell. On the basis of the present data we suggest a controlling role of mitochondrial LDH regarding extramitochondrial oxidation of NADH in the presence of cytochrome c, via rotenone-insensitive NADH-cytochrome c reductase. Extramitochondrial oxidation of NADH via an “external” pathway has already been described (Sottocasa et al., 1967; Bernardi and Azzone, 1981; Bernardi and Azzone, 1982; Szczesna-Kaczmarek et al., 1984), but with regard to energy conservation (only 1 side of ATP synthesis, Bernardi and Azzone, 1982), the role of this pathway is still unclear. We suggested previously that the skeletal cell utilizes this uneconomical system only exclusively as a defence mechanism against the effect of prolonged acidosis caused by accumulation of lactic acid (Szczesna-Kaczmarek et al., 1984). In the conditions mentioned above such a form of respiration acts as a hydrogen transporting shuttle, At present we have shown the mechanism controlling the activity of the “external” pathway acting as a shuttle transporting reducing equivalents. This report shows that pyruvate exerts an inhibitory effect on external
NADH oxidation in the presence of cytochrome c due to the presence of mitochondrial LDH. The reduction rate of pyruvate to lactate was sufficient to keep extramitochondrial NADH and hinder oxidation through the “external” pathway. It may be concluded that mitochondrial lactate dehydrogenase successfully competes with an “external” pathway for cytoplasmic NAD until the lactate/pyruvate ratio reaches a value allowing extramitochondrial NADH to act as a substrate for rotenone-insensitive NADHcytochrome c reductase. REFERENCES
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