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The effect of phosphoenolypyruvate on calcium transport by mitochondria
Biochimica et Biophysica Acta, 307 (1973) 599-606 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 76323 THE E F F E C T OF P H O S P H O E N O L P Y R U V A T E ON C A L C I U M T R A N S P O R T BY M I T O C H O N D R I A PRAKORN CHUDAPONGSE and NIELS HAUGAARD Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.) (Received December 8th, 1972)
SUMMARY Phosphoenolpyruvate was found to inhibit net uptake of Ca 2 + by rat heart and liver mitochondria. The main action of phosphoenolpyruvate is to increase the rate of efflux of mitochondrial Ca z +. The effect of phosphoenolpyruvate on mitochondrial Ca z+ transport is antagonized by ATP and by atractylate and is observed when mitochondria are respiring in the presence of NAD-linked substrates such as glutamate and pyruvate plus malate. In liver mitochondria phosphoenolpyruvate is also effective in the presence of succinate but not when rotenone is added. Glycolytic intermediates other than phosphoenolpyruvate had little effect on mitochondrial Ca 2 + transport.
INTRODUCTION in a study of the action of various glycolytic intermediates on mitocbondrial Ca 2+ transport we observed that phosphoenolpyruvate inhibited Ca 2+ uptake by heart mitochondria incubated in vitro but that other glycolytic intermediates studied had little or no effect on this process 1. McCoy and Doeg 2 have reported that phosphoenolpyruvate also inhibited in vitro protein synthesis by liver mitochondria. In the present publication we present results of experiments on the action of phosphoenolpyruvate on Ca 2+ transport by liver and heart mitochondria which show that the main action of phosphoenolpyruvate is to produce Ca 2 + efflux in the presence of respiratory substrates linked to the reduction of NAD. METHODS Preparation of mitochondria Mitochondria were prepared from rat hearts by the method of Von Korff 3. The final suspension of mitochondria was in 0.18 M KC1. Rat liver mitochondria in 0.250 M sucrose were prepared Soy the method of Hogeboom4 as described by Myers and Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-etbanesulfonic acid; TMPD, tetramethylphenylenediamine.
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Slater s. The protein concentrations were determined with biuret according to Cleland and Slater 6.
Incubation Mitochondria suspended in the appropriate reaction media were incubated at 26 °C in small beakers stirred with rotating magnets and open to the air. The total volumes of the mixtures were 4-5 ml and 1-ml samples were removed at timed intervals and filtered through Millipore filters with pore diameters of 0.45 or 0.65 #m. Preparation of Ca 2 +-loaded mitochondria In experiments in which Ca z + extrusion was followed, heart mitochondria were preloaded with Ca 2+ as follows: freshly prepared mitochondria (3 4 ml) were added to 10 ml solution containing 0.040 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 0.010 M MgCl 2 and 0.080 M KC1.2 ml 0.1 M sodium glutamate, 1.5 ml 0.02 M potassium phosphate and 1 ml 0.02 M CaCI 2 were then added and the mixture incubated with stirring at 26 °C for 5 rain. The mitochondria were removed by centrifugation at 12000 x g for 5 min and washed twice with 0.18 M KCI by resuspension and centrifugation. They were finally suspended in 0.18 M KCI by gentle homogenization. Although the Ca 2+ concentration (1.1 raM) in the loading experiments was high, the mitochondrial content was twice as high as in the other experiments so that the amount of Ca 2 + per mg of mitochondria was about the same in all experiments performed. Determination of Ca 2 + The solutions obtained after Millipore filtration were diluted about 1/12 with 0.1 M KC1-0.01 M SrCI2 and the Ca 2+ concentrations determined by atomic absorption spectroscopy (Perkin-Elmer Model 290B). Chemicals HEPES, dithiothreitol and phosphoenolpyruvate (as the potassium or tricyclohexylammonium salt) were obtained from Calbiochem and oligomycin from Sigma Chemical Co. Atractylate was a gift from Dr R. Santi. RESULTS
Effect of phosphoenolpyruvate on respiration-supported calcium uptake by rat heart mitochondria Experimental results illustrating the effect of phosphoenolpyruvate on Ca z + uptake by heart mitochondria in the presence of glutamate and inorganic phosphate are recorded in Fig. 1. In these experiments phosphoenolpyruvate was added 1 rain before CaC1 z. A significant inhibition of Ca 2 + uptake was observed when as little as 0.12 m M phosphoenolpyruvate was present and the degree of inhibition increased as the concentration of phosphoenolpyruvate was raised. It is important to note that the initial rate of Ca 2+ uptake was little affected and that the inhibition of net uptake of Ca 2+ increased as the incubation proceeded. This was substantiated in a similar experiment in which the Ca 2 + concentration in the medium was determined at 30-s intervals. In
CALCIUM TRANSPORT BY MITOCHONDRIA
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other experiments it was observed that the i n h i b i t o r y effects of small c o n c e n t r a t i o n s of p h o s p h o e n o l p y r u v a t e were e n h a n c e d when the m i t o c h o n d r i a were pre-exposed to p h o s p h o e n o l p y r u v a t e for more t h a n 1 rain. Similar results were o b t a i n e d with the p o t a s s i u m and t r i c y c l o h e x y l a m m o n i u m salts of p h o s p h o e n o l p y r u v a t e . Other glycolytic intermediates were also tested. Fructose 1,6-diphosphate, glucose 6-phosphate a n d glucose 1-phosphate were f o u n d to be ineffective at concent r a t i o n s as high as 2.4 m M . A small b u t significant i n h i b i t i o n was observed with 1.0 m M 3-phosphoglyceric acid, possible caused by conversion of this substance to p h o s p h o e n o l p y r u v a t e by glycolytic enzymes present in the m i t o c h o n d r i a l preparations.
Reversal of the effect of phosphoenolpyruvate by A TP and atractylate Experimental results showing the a n t a g o n i s t i c actions of A T P and atractylate on the effect of p h o s p h o e n o l p y r u v a t e on Ca/+ uptake by heart m i t o c h o n d r i a are recorded in Fig. 2. In these experiments the m i t o c h o n d r i a were p r e i n c u b a t e d for 1 min before a d d i t i o n of CaCI z. ATP, p h o s p h o e n o l p y r u v a t e or atractylate were added initially as indicated. It is seen that 0.47 m M A T P completely overcame the i n h i b i t o r y effect of
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Fig. : 1 Effect of phosphoenolpyruvate on Ca 2+ uptake by rat heart mitochondria. Composition of reaction system: 29 mM HEPES (pH 7.4), 7.3 mM MgCI2, 7.3 mM sodium glutamate, KCI to 250 mosM, 0.5 mM potassium phosphate (pH 7.4) and phosphoenolpyruvate as indicated. 0.47 mM CaCI2 added after 1 rain of preincubation with and without phosphoenolpyruvate. 0.67 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4.1 ml. PEP, phosphoenolpyruvate. Fig. 2. Left panel. Reversal of action of phosphoenolpyruvate by ATP with rat heart mitochondria. Composition of reaction systems: 29 mM HEPES (pH 7.4), 7.2 mM MgClz, 7.2 mM sodium glutamate, KCI to 250 mosM, 0.72 mM potassium phosphate (pH 7.4), phosphoenolpyruvate and ATP as indicated. 0.45 mM CaC12 added after 1 rain of preincubation with and without phosphoenolpyruvate. ATP, when present, added at the same time as phosphoenolpyruvate. 0.94 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4.12 ml. Right panel. Reversal of the action of phosphoenolpyruvate by atractylate with rat heart mitochondria. Composition of reaction system 29 mM HEPES (pH 7.4), 7.2 mM MgC12, 7.2 mM sodium glutamate, KCI to 250 mosM. 0.72 mM potassium phosphate (pH 7.4), phosphoenolpyruvate and atractylate as indicated, 0.44 mM CaC12 added 1 rain after preincubation with or without inhibitors. When both phosphoenolpyruvate and atractylate were present they were added at the same time. 0.51 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4,19 ml. PEP, phosphoenolpyruvate; ATRAC., atractylate.
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1.2 m M p h o s p h o e n o l p y r u v a t e (left panel o f Fig. 2). In o t h e r experiments, it was shown t h a t o l i g o m y c i n or a t r a c t y l a t e did n o t interfere with the a c t i o n o f A T P in preventing the effect o f p h o s p h o e n o l p y r u v a t e on calcium uptake. In similar experiments it was o b s e r v e d t h a t ITP or I M P at c o n c e n t r a t i o n s o f 0.24 m M were t o t a l l y ineffective in o p p o s i n g the a c t i o n o f p h o s p h o e n o l p y r u v a t e . Results f r o m experiments on the a c t i o n o f a t r a c t y l a t e on heart m i t o c h o n d r i a i n c u b a t e d with g l u t a m a t e a n d p h o s p h a t e but no A T P are recorded in the right panel o f Fig. 2. It is seen t h a t 0.06 m M a t r a c t y l a t e c o m p l e t e l y overcame the effect o f 0.65 m M p h o s p h o e n o l p y r u v a t e . A c t u a l l y smaller c o n c e n t r a t i o n s o f a t r a c t y l a t e were equally effective since 6 # M o f the i n h i b i t o r also a b o l i s h e d the effect o f 0.65 m M p h o s p h o enolpyruvate. A T P itself is very p o t e n t in a n t a g o n i z i n g the a c t i o n of p h o s p h o e n o l p y r u v a t e as seen from the e x p e r i m e n t s r e p o r t e d in the left panel o f Fig. 3. CALCIUM
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Fig. 3. Left panel. Inhibition of the action phosphoenolpyruvate by different concentrations of ATP with rat heart mitochondria. Composition of reaction system: 29 mM HEPES (pH 7.4), 7.3 mM MgCIz, 7.3 mM sodium glutamate, KCI to 250 mosM, 0.73 mM potassium phosphate (pH 7.4) 1.22 mM phosphoenolpyruvate and ATP as indicated. CaCI2 (0.52 raM) added after 1 rain of preincubation with and without phosphoenolpyruvate. ATP, when present, added at the same time as phosphoenolpyruvate. 0.79 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4.1 ml. Right panel. Extrusion of Ca 2+ from Ca2+-Ioaded rat heart mitochondria. Composition of reaction system: 31 mM HEPES (pH 7.4), 7.7 mM MgCI2, 7.7 mM sodium glutamate, KCI to 250 mosM. When present, 1.2 mM phosphoenolpyruvate, 0.51 mM ATP or 41/~M dinitrophenol. Phosphoenolpyruvate (with and without ATP) and dinitrophenol were added after 1 rain preincubation. Mitochondria were loaded with Ca 2+ as described in Methods. 1.07 mg mitochondrial protein per ml. Temperature 26 "C. Total volume 3.9 ml. PEP, phosphoenolpyruvate; DNP, dinitrophenol. W h e n the i n c u b a t i o n s were carried o u t in the presence o f 1.22 m M p h o s p h o e n o l p y r u v a t e as little as 5 # M A T P had a significant effect in alleviating the i n h i b i t i o n caused by p h o s p h o e n o l p y r u v a t e and increasing c o n c e n t r a t i o n s o f A T P further overcame the a c t i o n o f p h o s p h o e n o l p y r u v a t e Effects o f phosphoenolpyruvate on Ca 2 + -loaded mitochondria The o b s e r v a t i o n t h a t p h o s p h o e n o l p y r u v a t e had little or no effect on the initial rate o f u p t a k e o f Ca 2+ but exerted its effect only after some m i t o c h o n d r i a l Ca 2+
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u p t a k e h a d t a k e n place suggested t h a t the a c t i o n o f p h o s p h o e n o l p y r u v a t e was t h a t o f increasing the rate o f efflux o f Ca 2+. To test this possibility, h e a r t m i t o c h o n d r i a p r e - l o a d e d with Ca 2+ as described in M e t h o d s were i n c u b a t e d in a m e d i u m c o n t a i n i n g g l u t a m a t e . In c o n t r o l s the C a / + c o n t e n t o f the m i t o c h o n d r i a was well m a i n t a i n e d but a d d i t i o n o f p h o s p h o e n o l p y r u v a t e caused extrusion of the a c c u m u l a t e d Ca 2+ (right panel of Fig. 3). The effect o f p h o s p h o e n o l p y r u v a t e on Ca 2 + efflux a l t h o u g h m a r k e d was slow in onset c o m p a r e d to the action o f 2 , 4 - d i n i t r o p h e n o l on this process. The a d J i t i o n o f A T P c o m p l e t e l y reversed the effect o f p h o s p h o e n o l p y r u v a t e .
Effect o f substrate on the action o[phoaphoenolpyruvate The e x p e r i m e n t s so far r e p o r t e d were carried out with heart m i t o c h o n d r i a i n c u b a t e d with g l u t a m a t e as the r e s p i r a t o r y substrate. The results o f experiments with different s u b s t r a t e s are r e p o r t e d in T a b l e I. TABLE I ROLE OF SUBSTRATE IN THE INHIBITORY EFFECT OF PHOSPHOENOLPYRUVATE ON Ca `-'+ UPTAKE BY RAT HEART M1TOCHONDRIA Composition of reaction system: 30 mM HEPES (pH 7.4), 7.4 mM MgCIz, 0.50 mM potassium phosphate, KCI to 250 mosM. When present, 1.22 mM phosphoenolpyruvate, 7.4 mM sodium glutamate, 3.7 mM sodium malate, 3.7 mM sodium pyruvate, 7.4 mM sodium succinate, 3.7 mM ascorbate and 0.250 mM TMPD. CaC12 (0.51 raM) added after 1 min of preincubation with and without phosphoenolpyruvate. 1.28 mg mitochondrial protein per ml. Temperature 26 PC. Total volume 4.05 ml.
Substrate
% inhibition at 3 min
% inhibition at 5 min
Glutamate Succinate Malate + pyruvate Ascorbate + TM PD
77 0 28 6
94 2 64 9.5
It is seen t h a t the degree o f i n h i b i t i o n o f net u p t a k e o f C a 2+ was large w i t h g l u t a m a t e as substrate, s o m e w h a t s m a l l e r with m a l a t e plus p y r u v a t e and a l m o s t a b s e n t with succinate or ascorbateplus t e t r a m e t h y l p h e n y l e n e d i a m i n e ( T M P D ) . These findings indicate t h a t the a c t i o n o f p h o s p h o e n o l p y r u v a t e involves r e a c t i o n s concerned with the first energy-conserving site o f the r e s p i r a t o r y chain.
Effect of phosphoenolpyruvate on Ca 2 + uptake by liver mitochondria Results o f e x p e r i m e n t s on the effect o f p h o s p h o e n o l p y r u v a t e on Ca z+ u p t a k e by rat liver m i t o c h o n d r i a are r e p o r t e d in T a b l e II. As with h e a r t m i t o c h o n d r i a , p h o s p h o e n o l p y r u v a t e m a r k e d l y i n h i b i t e d net C a 2+ u p t a k e in the presence o f glutam a t e a n d p h o s p h a t e . H o w e v e r , in c o n t r a s t to the findings with h e a r t m i t o c h o n d r i a , Ca 2+ u p t a k e was also s t r o n g l y i n h i b i t e d when succinate was the r e s p i r a t o r y substrate. W e then carried o u t similar e x p e r i m e n t s with r o t e n o n e present. U n d e r these conditions, when e n d o g e n o u s N A D H o x i d a t i o n was inhibited, p h o s p h o e n o l p y r u v a t e had
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no effect on mitochondrial calcium uptake (Table lI) again indicating that the action of phosphoenolpyruvate involves reactions in the early part of the electron transport chain. TABLE I1 THE E F F E C T OF P H O S P H O E N O L P Y R U V A T E ON Ca 2+ U P T A K E BY RAT LIVER
MITOCHONDRIA Composition of reaction system: 30 m M HEPES (pH 7.4), 7.5 mM MgCI2, 7.5 mM sodium glutamate or succinate, 0.75 mM potassium phosphate, KCI to 250 mosM, 0.030 M sucrose. Phospho-
enolpyruvate concentration as indicated. CaClz added after 1 rain preincubation (0.44 mM in Expt 1, 0.47 mM in Expt 2). Rotenone 5.0/~g/ml, when present. Temperature 26 ~'C. Mitochondrial protein 1.16 mg/ml in Expt l, 1.48 mg/ml in Expt 2. Total volume 4.15 ml in Expt 1, 4.04 ml in Expt 2. Expt No.
Substrate
Phosphoenolpyruvate ( m M )
Ca 2 + uptake
(Itmoles/ml)
1 rain
5 min
l
Glutamate + Pl
0 0.60
0.281 0.220
0.389 0.121
2
Succinate + Pl Succinate + PI + rotenone
0 0.67 0 0.67
0.320 0.144 0.458 0.458
0.446 0.091 0.458 0.458
It is interesting that rotenone markedly increased C a 2 + uptake by liver mitochondria respiring in the presence of succinate. This is general phenomenon that we have observed consistently in many experiments and may be related to inhibition of malate oxidation by rotenone. DISCUSSION
The results presented here show that an intermediate of glycolysis can influence an important cellular function of mitochondria, the uptake and release of Ca z+. Phosphoenolpyruvate appears to be unique in this respect, since other glycolytic intermediates had little or no effect on mitochondrial Ca 2+ transport. The mechanism of the effect of phosphoenolpyruvate on mitochondrial Ca 2+ transport is not clear at present. One possibility that we considered was that phosphoenolpyruvate acts as a substrate for the pyruvate kinase reaction, phosphoenolpyruv a t e + A D P = P y r u v a t e + A T P , and in this way removes A D P present between the inner and outer mitochondrial membranes. This does not happen since in experiments with heart mitochondria incubated in vitro we found that phosphoenolpyruvate was not metabolized during a 3-rain incubation even in the presence of excess ADP. Another possibility is that phosphoenolpyruvate inhibits the oxidation of mitochondrial N A D H . However, experiments with liver mitochondria exposed to an osmotic shock (five times dilution of the original suspension with water) gave no support to this hypothesis since phosphoenolpyruvate in concentrations up to 5 mM caused a slight increase rather than a decrease in the rate of oxidation of added N A D H . With intact liver mitochondria phosphoenolpyruvate does not influence State 3 or State 4
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respiration and inhibits 2,4-dinitrophenol-stimulated respiration in the presence of glutamate to only a small extent. The fact that the effects of phosphoenolpyruvate described here are seen when the mitochondria are respiring in the presence of substrates giving rise to the formation of N A D H suggests that the mechanisms of the action of phosphoenolpyruvate involves an interaction with the process of energy conservation at Site I. Since atractylate and ATP are so effective in overcoming the effects of phosphoenolpyruvate, this compound appears to combine with the same site as atractylate and ATP, presumably the adenine nucleotide translocator on the inner mitochondrial membrane. Phosphoenolpyruvate most probably does not exert its action intramitochondrially since the tricarboxylate translocator, which is involved in transport of phosphoenolpyruvate across the inner mitochondrial membrane 8, is absent in heart mitochondria 9. The effectiveness of atractylate in opposing the action of phosphoenolpyruvate also supports this conclusion. Phosphoenolpyruvate does not appear to act by interfering with phosphate transport since dithiodinitrobenzoic acid, a potent inhibitor of phosphate uptake by mitochondria 7, does not cause extrusion of Ca 2+ from mitochondria and increasing the phosphate concentration of the medium did not diminish the action of phosphoenolpyruvate. Vignais e t al. t° have proposed that atractylate, its aglycone, atractylegenin, and other substances with similar action produce their effects by combining with an allosteric effector site that is part of the adenine nucleotide translocator. One can speculate that phosphoenolpyruvate also acts in a similar manner. It should be noted in this connection that phosphoenolpyruvate contains a methylene group, a structure in the atractylate molecule necessary for full activity of this compound ~1 The observation of McCoy and Doeg 2 that mitochondrial protein synthesis is inhibited by phosphoenolpyruvate indicates that phosphoenolpyruvate may influence several mitochondrial reactions. The inhibition of protein synthesis reported by these authors was found at somewhat higher concentrations of phosphoenolpyruvate than the effects observed here but in both cases the action of phosphoenolpyruvate was overcome by atractylate or ATP indicating that the same site of action may be involved. Whatever the mechanism of action of phosphoenolpyruvate the most important aspect of the problem is the possibility that changes in glycolytic flux can influence mitochondrial reactions in the intact cell. For phosphoenolpyruvate to be effective in doing so it must reach the mitochondrion at a time which the ATP concentration in the immediate vicinity is low. Further experimentation is needed to determine whether changes in the concentration of phosphoenolpyruvate could play a physiological role in the regulation of mitochondrial metabolism and function. ACKNOWLEDGMENTS These studies were supported by a grant from the National Institutes of Health, U. S. Public Health Service (HL-01813). P. C. was supported by a predoctoral fellowship from the Rockefeller Foundation. The experimental data presented here are taken from a thesis by P. C. in partial fulfillment of the degree of P h . D . in Pharmacology at the University of Pennsylvania. We thank Professor R. Santi for his generous gift of a sample of atractylate used in this work.
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REFERENCES 1 2 3 4 5 6 7 8 9 10 11
Chudapongse, P. and Haugaard, N. (1972) Fed. Proc. 31,849 McCoy, E. D. and Doeg, K. A. (1972) Biochem. Biophys. Res. Cornrnun. 46, 1411-1417 Von Korff, R. W. (1965) J. Biol. Chem. 240, 1351-1364 Hogeboom, E. H. (1955) in Methods" in Enzyrnology (Colowick, S. P. and Kaplan, N. O., eds), Vol. 1, pp. 16 18, Academic Press, New York Myers, D. K. and S|ater, E. C. (1957) Biochem. J. 67, 558-572 Cleland, K. W. and Slater, E. C. (1953) Biochem. J. 53, 547-556 Haugaard, N., Lee, H. H., Kostrzewa, R., Horn, R. S. and Haugaard, E. (1969) Biochim. Biophys. Acta 172, 198-204 Gamble, Jr, J. L. and Mazur, J. A. (1967) J. BioL Chem. 242, 67-72 Chappell, J. B. (1966) Biochem. J. 100, 43P Vignais, P. V., Duee, E. D., Vignais, P. M., and Huet, J. (1966) Biochirn. Biophys. Acta 118, 465-483 Santi, R., Bruni, A., Contessa, A. R. and Luciani, S. (1962) Boll. Soc. ltal. Biol. Sper. 38, 18901891
SUMMARY Phosphoenolpyruvate was found to inhibit net uptake of Ca 2 + by rat heart and liver mitochondria. The main action of phosphoenolpyruvate is to increase the rate of efflux of mitochondrial Ca z +. The effect of phosphoenolpyruvate on mitochondrial Ca z+ transport is antagonized by ATP and by atractylate and is observed when mitochondria are respiring in the presence of NAD-linked substrates such as glutamate and pyruvate plus malate. In liver mitochondria phosphoenolpyruvate is also effective in the presence of succinate but not when rotenone is added. Glycolytic intermediates other than phosphoenolpyruvate had little effect on mitochondrial Ca 2 + transport.
INTRODUCTION in a study of the action of various glycolytic intermediates on mitocbondrial Ca 2+ transport we observed that phosphoenolpyruvate inhibited Ca 2+ uptake by heart mitochondria incubated in vitro but that other glycolytic intermediates studied had little or no effect on this process 1. McCoy and Doeg 2 have reported that phosphoenolpyruvate also inhibited in vitro protein synthesis by liver mitochondria. In the present publication we present results of experiments on the action of phosphoenolpyruvate on Ca 2+ transport by liver and heart mitochondria which show that the main action of phosphoenolpyruvate is to produce Ca 2 + efflux in the presence of respiratory substrates linked to the reduction of NAD. METHODS Preparation of mitochondria Mitochondria were prepared from rat hearts by the method of Von Korff 3. The final suspension of mitochondria was in 0.18 M KC1. Rat liver mitochondria in 0.250 M sucrose were prepared Soy the method of Hogeboom4 as described by Myers and Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-etbanesulfonic acid; TMPD, tetramethylphenylenediamine.
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Slater s. The protein concentrations were determined with biuret according to Cleland and Slater 6.
Incubation Mitochondria suspended in the appropriate reaction media were incubated at 26 °C in small beakers stirred with rotating magnets and open to the air. The total volumes of the mixtures were 4-5 ml and 1-ml samples were removed at timed intervals and filtered through Millipore filters with pore diameters of 0.45 or 0.65 #m. Preparation of Ca 2 +-loaded mitochondria In experiments in which Ca z + extrusion was followed, heart mitochondria were preloaded with Ca 2+ as follows: freshly prepared mitochondria (3 4 ml) were added to 10 ml solution containing 0.040 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 0.010 M MgCl 2 and 0.080 M KC1.2 ml 0.1 M sodium glutamate, 1.5 ml 0.02 M potassium phosphate and 1 ml 0.02 M CaCI 2 were then added and the mixture incubated with stirring at 26 °C for 5 rain. The mitochondria were removed by centrifugation at 12000 x g for 5 min and washed twice with 0.18 M KCI by resuspension and centrifugation. They were finally suspended in 0.18 M KCI by gentle homogenization. Although the Ca 2+ concentration (1.1 raM) in the loading experiments was high, the mitochondrial content was twice as high as in the other experiments so that the amount of Ca 2 + per mg of mitochondria was about the same in all experiments performed. Determination of Ca 2 + The solutions obtained after Millipore filtration were diluted about 1/12 with 0.1 M KC1-0.01 M SrCI2 and the Ca 2+ concentrations determined by atomic absorption spectroscopy (Perkin-Elmer Model 290B). Chemicals HEPES, dithiothreitol and phosphoenolpyruvate (as the potassium or tricyclohexylammonium salt) were obtained from Calbiochem and oligomycin from Sigma Chemical Co. Atractylate was a gift from Dr R. Santi. RESULTS
Effect of phosphoenolpyruvate on respiration-supported calcium uptake by rat heart mitochondria Experimental results illustrating the effect of phosphoenolpyruvate on Ca z + uptake by heart mitochondria in the presence of glutamate and inorganic phosphate are recorded in Fig. 1. In these experiments phosphoenolpyruvate was added 1 rain before CaC1 z. A significant inhibition of Ca 2 + uptake was observed when as little as 0.12 m M phosphoenolpyruvate was present and the degree of inhibition increased as the concentration of phosphoenolpyruvate was raised. It is important to note that the initial rate of Ca 2+ uptake was little affected and that the inhibition of net uptake of Ca 2+ increased as the incubation proceeded. This was substantiated in a similar experiment in which the Ca 2 + concentration in the medium was determined at 30-s intervals. In
CALCIUM TRANSPORT BY MITOCHONDRIA
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other experiments it was observed that the i n h i b i t o r y effects of small c o n c e n t r a t i o n s of p h o s p h o e n o l p y r u v a t e were e n h a n c e d when the m i t o c h o n d r i a were pre-exposed to p h o s p h o e n o l p y r u v a t e for more t h a n 1 rain. Similar results were o b t a i n e d with the p o t a s s i u m and t r i c y c l o h e x y l a m m o n i u m salts of p h o s p h o e n o l p y r u v a t e . Other glycolytic intermediates were also tested. Fructose 1,6-diphosphate, glucose 6-phosphate a n d glucose 1-phosphate were f o u n d to be ineffective at concent r a t i o n s as high as 2.4 m M . A small b u t significant i n h i b i t i o n was observed with 1.0 m M 3-phosphoglyceric acid, possible caused by conversion of this substance to p h o s p h o e n o l p y r u v a t e by glycolytic enzymes present in the m i t o c h o n d r i a l preparations.
Reversal of the effect of phosphoenolpyruvate by A TP and atractylate Experimental results showing the a n t a g o n i s t i c actions of A T P and atractylate on the effect of p h o s p h o e n o l p y r u v a t e on Ca/+ uptake by heart m i t o c h o n d r i a are recorded in Fig. 2. In these experiments the m i t o c h o n d r i a were p r e i n c u b a t e d for 1 min before a d d i t i o n of CaCI z. ATP, p h o s p h o e n o l p y r u v a t e or atractylate were added initially as indicated. It is seen that 0.47 m M A T P completely overcame the i n h i b i t o r y effect of
10.6
O6
0.5 0.5
0.2
u 0.1
0.5
0.4
,~ 0,4 ,'7 02 _z
I
0.6
~
0.3 0.0488 mM PEP CONTROL ~
/,~_~0 2
t0 I
J MINUTES
\ ~e----"~
,.J ~. o.3
PEP I0.3
~ 0.2 ~
uO.I
mM PEP ~ +O,47mM ATP
0.65mM PEI +AT RAC.
~bO'l ~
CONTROL / ATRAC.
i
t
t
i
I
I
I
I
I
I
2
3
4
0
I
2
3
4
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MINUTES
Fig. : 1 Effect of phosphoenolpyruvate on Ca 2+ uptake by rat heart mitochondria. Composition of reaction system: 29 mM HEPES (pH 7.4), 7.3 mM MgCI2, 7.3 mM sodium glutamate, KCI to 250 mosM, 0.5 mM potassium phosphate (pH 7.4) and phosphoenolpyruvate as indicated. 0.47 mM CaCI2 added after 1 rain of preincubation with and without phosphoenolpyruvate. 0.67 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4.1 ml. PEP, phosphoenolpyruvate. Fig. 2. Left panel. Reversal of action of phosphoenolpyruvate by ATP with rat heart mitochondria. Composition of reaction systems: 29 mM HEPES (pH 7.4), 7.2 mM MgClz, 7.2 mM sodium glutamate, KCI to 250 mosM, 0.72 mM potassium phosphate (pH 7.4), phosphoenolpyruvate and ATP as indicated. 0.45 mM CaC12 added after 1 rain of preincubation with and without phosphoenolpyruvate. ATP, when present, added at the same time as phosphoenolpyruvate. 0.94 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4.12 ml. Right panel. Reversal of the action of phosphoenolpyruvate by atractylate with rat heart mitochondria. Composition of reaction system 29 mM HEPES (pH 7.4), 7.2 mM MgC12, 7.2 mM sodium glutamate, KCI to 250 mosM. 0.72 mM potassium phosphate (pH 7.4), phosphoenolpyruvate and atractylate as indicated, 0.44 mM CaC12 added 1 rain after preincubation with or without inhibitors. When both phosphoenolpyruvate and atractylate were present they were added at the same time. 0.51 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4,19 ml. PEP, phosphoenolpyruvate; ATRAC., atractylate.
602
P. CHUDAPONGSE, N. HAUGAARD
1.2 m M p h o s p h o e n o l p y r u v a t e (left panel o f Fig. 2). In o t h e r experiments, it was shown t h a t o l i g o m y c i n or a t r a c t y l a t e did n o t interfere with the a c t i o n o f A T P in preventing the effect o f p h o s p h o e n o l p y r u v a t e on calcium uptake. In similar experiments it was o b s e r v e d t h a t ITP or I M P at c o n c e n t r a t i o n s o f 0.24 m M were t o t a l l y ineffective in o p p o s i n g the a c t i o n o f p h o s p h o e n o l p y r u v a t e . Results f r o m experiments on the a c t i o n o f a t r a c t y l a t e on heart m i t o c h o n d r i a i n c u b a t e d with g l u t a m a t e a n d p h o s p h a t e but no A T P are recorded in the right panel o f Fig. 2. It is seen t h a t 0.06 m M a t r a c t y l a t e c o m p l e t e l y overcame the effect o f 0.65 m M p h o s p h o e n o l p y r u v a t e . A c t u a l l y smaller c o n c e n t r a t i o n s o f a t r a c t y l a t e were equally effective since 6 # M o f the i n h i b i t o r also a b o l i s h e d the effect o f 0.65 m M p h o s p h o enolpyruvate. A T P itself is very p o t e n t in a n t a g o n i z i n g the a c t i o n of p h o s p h o e n o l p y r u v a t e as seen from the e x p e r i m e n t s r e p o r t e d in the left panel o f Fig. 3. CALCIUM
0.6
0.5'
UPTAKE
0.5
~2 .~ 0.4 z
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0 ~?
E
u_
CALCIUM EXTRUSION
~_
0.3
0.4 0.3
DNP
~ 0.2
0.2
PEP
PEP+ATPJ ~ i
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2 3 MINUTES
i
4
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i 2
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Fig. 3. Left panel. Inhibition of the action phosphoenolpyruvate by different concentrations of ATP with rat heart mitochondria. Composition of reaction system: 29 mM HEPES (pH 7.4), 7.3 mM MgCIz, 7.3 mM sodium glutamate, KCI to 250 mosM, 0.73 mM potassium phosphate (pH 7.4) 1.22 mM phosphoenolpyruvate and ATP as indicated. CaCI2 (0.52 raM) added after 1 rain of preincubation with and without phosphoenolpyruvate. ATP, when present, added at the same time as phosphoenolpyruvate. 0.79 mg mitochondrial protein per ml. Temperature 26 °C. Total volume 4.1 ml. Right panel. Extrusion of Ca 2+ from Ca2+-Ioaded rat heart mitochondria. Composition of reaction system: 31 mM HEPES (pH 7.4), 7.7 mM MgCI2, 7.7 mM sodium glutamate, KCI to 250 mosM. When present, 1.2 mM phosphoenolpyruvate, 0.51 mM ATP or 41/~M dinitrophenol. Phosphoenolpyruvate (with and without ATP) and dinitrophenol were added after 1 rain preincubation. Mitochondria were loaded with Ca 2+ as described in Methods. 1.07 mg mitochondrial protein per ml. Temperature 26 "C. Total volume 3.9 ml. PEP, phosphoenolpyruvate; DNP, dinitrophenol. W h e n the i n c u b a t i o n s were carried o u t in the presence o f 1.22 m M p h o s p h o e n o l p y r u v a t e as little as 5 # M A T P had a significant effect in alleviating the i n h i b i t i o n caused by p h o s p h o e n o l p y r u v a t e and increasing c o n c e n t r a t i o n s o f A T P further overcame the a c t i o n o f p h o s p h o e n o l p y r u v a t e Effects o f phosphoenolpyruvate on Ca 2 + -loaded mitochondria The o b s e r v a t i o n t h a t p h o s p h o e n o l p y r u v a t e had little or no effect on the initial rate o f u p t a k e o f Ca 2+ but exerted its effect only after some m i t o c h o n d r i a l Ca 2+
CALCIUM TRANSPORT BY MITOCHONDRIA
603
u p t a k e h a d t a k e n place suggested t h a t the a c t i o n o f p h o s p h o e n o l p y r u v a t e was t h a t o f increasing the rate o f efflux o f Ca 2+. To test this possibility, h e a r t m i t o c h o n d r i a p r e - l o a d e d with Ca 2+ as described in M e t h o d s were i n c u b a t e d in a m e d i u m c o n t a i n i n g g l u t a m a t e . In c o n t r o l s the C a / + c o n t e n t o f the m i t o c h o n d r i a was well m a i n t a i n e d but a d d i t i o n o f p h o s p h o e n o l p y r u v a t e caused extrusion of the a c c u m u l a t e d Ca 2+ (right panel of Fig. 3). The effect o f p h o s p h o e n o l p y r u v a t e on Ca 2 + efflux a l t h o u g h m a r k e d was slow in onset c o m p a r e d to the action o f 2 , 4 - d i n i t r o p h e n o l on this process. The a d J i t i o n o f A T P c o m p l e t e l y reversed the effect o f p h o s p h o e n o l p y r u v a t e .
Effect o f substrate on the action o[phoaphoenolpyruvate The e x p e r i m e n t s so far r e p o r t e d were carried out with heart m i t o c h o n d r i a i n c u b a t e d with g l u t a m a t e as the r e s p i r a t o r y substrate. The results o f experiments with different s u b s t r a t e s are r e p o r t e d in T a b l e I. TABLE I ROLE OF SUBSTRATE IN THE INHIBITORY EFFECT OF PHOSPHOENOLPYRUVATE ON Ca `-'+ UPTAKE BY RAT HEART M1TOCHONDRIA Composition of reaction system: 30 mM HEPES (pH 7.4), 7.4 mM MgCIz, 0.50 mM potassium phosphate, KCI to 250 mosM. When present, 1.22 mM phosphoenolpyruvate, 7.4 mM sodium glutamate, 3.7 mM sodium malate, 3.7 mM sodium pyruvate, 7.4 mM sodium succinate, 3.7 mM ascorbate and 0.250 mM TMPD. CaC12 (0.51 raM) added after 1 min of preincubation with and without phosphoenolpyruvate. 1.28 mg mitochondrial protein per ml. Temperature 26 PC. Total volume 4.05 ml.
Substrate
% inhibition at 3 min
% inhibition at 5 min
Glutamate Succinate Malate + pyruvate Ascorbate + TM PD
77 0 28 6
94 2 64 9.5
It is seen t h a t the degree o f i n h i b i t i o n o f net u p t a k e o f C a 2+ was large w i t h g l u t a m a t e as substrate, s o m e w h a t s m a l l e r with m a l a t e plus p y r u v a t e and a l m o s t a b s e n t with succinate or ascorbateplus t e t r a m e t h y l p h e n y l e n e d i a m i n e ( T M P D ) . These findings indicate t h a t the a c t i o n o f p h o s p h o e n o l p y r u v a t e involves r e a c t i o n s concerned with the first energy-conserving site o f the r e s p i r a t o r y chain.
Effect of phosphoenolpyruvate on Ca 2 + uptake by liver mitochondria Results o f e x p e r i m e n t s on the effect o f p h o s p h o e n o l p y r u v a t e on Ca z+ u p t a k e by rat liver m i t o c h o n d r i a are r e p o r t e d in T a b l e II. As with h e a r t m i t o c h o n d r i a , p h o s p h o e n o l p y r u v a t e m a r k e d l y i n h i b i t e d net C a 2+ u p t a k e in the presence o f glutam a t e a n d p h o s p h a t e . H o w e v e r , in c o n t r a s t to the findings with h e a r t m i t o c h o n d r i a , Ca 2+ u p t a k e was also s t r o n g l y i n h i b i t e d when succinate was the r e s p i r a t o r y substrate. W e then carried o u t similar e x p e r i m e n t s with r o t e n o n e present. U n d e r these conditions, when e n d o g e n o u s N A D H o x i d a t i o n was inhibited, p h o s p h o e n o l p y r u v a t e had
604
P. C H U D A P O N G S E , N. H A U G A A R D
no effect on mitochondrial calcium uptake (Table lI) again indicating that the action of phosphoenolpyruvate involves reactions in the early part of the electron transport chain. TABLE I1 THE E F F E C T OF P H O S P H O E N O L P Y R U V A T E ON Ca 2+ U P T A K E BY RAT LIVER
MITOCHONDRIA Composition of reaction system: 30 m M HEPES (pH 7.4), 7.5 mM MgCI2, 7.5 mM sodium glutamate or succinate, 0.75 mM potassium phosphate, KCI to 250 mosM, 0.030 M sucrose. Phospho-
enolpyruvate concentration as indicated. CaClz added after 1 rain preincubation (0.44 mM in Expt 1, 0.47 mM in Expt 2). Rotenone 5.0/~g/ml, when present. Temperature 26 ~'C. Mitochondrial protein 1.16 mg/ml in Expt l, 1.48 mg/ml in Expt 2. Total volume 4.15 ml in Expt 1, 4.04 ml in Expt 2. Expt No.
Substrate
Phosphoenolpyruvate ( m M )
Ca 2 + uptake
(Itmoles/ml)
1 rain
5 min
l
Glutamate + Pl
0 0.60
0.281 0.220
0.389 0.121
2
Succinate + Pl Succinate + PI + rotenone
0 0.67 0 0.67
0.320 0.144 0.458 0.458
0.446 0.091 0.458 0.458
It is interesting that rotenone markedly increased C a 2 + uptake by liver mitochondria respiring in the presence of succinate. This is general phenomenon that we have observed consistently in many experiments and may be related to inhibition of malate oxidation by rotenone. DISCUSSION
The results presented here show that an intermediate of glycolysis can influence an important cellular function of mitochondria, the uptake and release of Ca z+. Phosphoenolpyruvate appears to be unique in this respect, since other glycolytic intermediates had little or no effect on mitochondrial Ca 2+ transport. The mechanism of the effect of phosphoenolpyruvate on mitochondrial Ca 2+ transport is not clear at present. One possibility that we considered was that phosphoenolpyruvate acts as a substrate for the pyruvate kinase reaction, phosphoenolpyruv a t e + A D P = P y r u v a t e + A T P , and in this way removes A D P present between the inner and outer mitochondrial membranes. This does not happen since in experiments with heart mitochondria incubated in vitro we found that phosphoenolpyruvate was not metabolized during a 3-rain incubation even in the presence of excess ADP. Another possibility is that phosphoenolpyruvate inhibits the oxidation of mitochondrial N A D H . However, experiments with liver mitochondria exposed to an osmotic shock (five times dilution of the original suspension with water) gave no support to this hypothesis since phosphoenolpyruvate in concentrations up to 5 mM caused a slight increase rather than a decrease in the rate of oxidation of added N A D H . With intact liver mitochondria phosphoenolpyruvate does not influence State 3 or State 4
CALCIUM TRANSPORT BY MITOCHONDRIA
605
respiration and inhibits 2,4-dinitrophenol-stimulated respiration in the presence of glutamate to only a small extent. The fact that the effects of phosphoenolpyruvate described here are seen when the mitochondria are respiring in the presence of substrates giving rise to the formation of N A D H suggests that the mechanisms of the action of phosphoenolpyruvate involves an interaction with the process of energy conservation at Site I. Since atractylate and ATP are so effective in overcoming the effects of phosphoenolpyruvate, this compound appears to combine with the same site as atractylate and ATP, presumably the adenine nucleotide translocator on the inner mitochondrial membrane. Phosphoenolpyruvate most probably does not exert its action intramitochondrially since the tricarboxylate translocator, which is involved in transport of phosphoenolpyruvate across the inner mitochondrial membrane 8, is absent in heart mitochondria 9. The effectiveness of atractylate in opposing the action of phosphoenolpyruvate also supports this conclusion. Phosphoenolpyruvate does not appear to act by interfering with phosphate transport since dithiodinitrobenzoic acid, a potent inhibitor of phosphate uptake by mitochondria 7, does not cause extrusion of Ca 2+ from mitochondria and increasing the phosphate concentration of the medium did not diminish the action of phosphoenolpyruvate. Vignais e t al. t° have proposed that atractylate, its aglycone, atractylegenin, and other substances with similar action produce their effects by combining with an allosteric effector site that is part of the adenine nucleotide translocator. One can speculate that phosphoenolpyruvate also acts in a similar manner. It should be noted in this connection that phosphoenolpyruvate contains a methylene group, a structure in the atractylate molecule necessary for full activity of this compound ~1 The observation of McCoy and Doeg 2 that mitochondrial protein synthesis is inhibited by phosphoenolpyruvate indicates that phosphoenolpyruvate may influence several mitochondrial reactions. The inhibition of protein synthesis reported by these authors was found at somewhat higher concentrations of phosphoenolpyruvate than the effects observed here but in both cases the action of phosphoenolpyruvate was overcome by atractylate or ATP indicating that the same site of action may be involved. Whatever the mechanism of action of phosphoenolpyruvate the most important aspect of the problem is the possibility that changes in glycolytic flux can influence mitochondrial reactions in the intact cell. For phosphoenolpyruvate to be effective in doing so it must reach the mitochondrion at a time which the ATP concentration in the immediate vicinity is low. Further experimentation is needed to determine whether changes in the concentration of phosphoenolpyruvate could play a physiological role in the regulation of mitochondrial metabolism and function. ACKNOWLEDGMENTS These studies were supported by a grant from the National Institutes of Health, U. S. Public Health Service (HL-01813). P. C. was supported by a predoctoral fellowship from the Rockefeller Foundation. The experimental data presented here are taken from a thesis by P. C. in partial fulfillment of the degree of P h . D . in Pharmacology at the University of Pennsylvania. We thank Professor R. Santi for his generous gift of a sample of atractylate used in this work.
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P. C H U D A P O N G S E , N. H A U G A A R D
REFERENCES 1 2 3 4 5 6 7 8 9 10 11
Chudapongse, P. and Haugaard, N. (1972) Fed. Proc. 31,849 McCoy, E. D. and Doeg, K. A. (1972) Biochem. Biophys. Res. Cornrnun. 46, 1411-1417 Von Korff, R. W. (1965) J. Biol. Chem. 240, 1351-1364 Hogeboom, E. H. (1955) in Methods" in Enzyrnology (Colowick, S. P. and Kaplan, N. O., eds), Vol. 1, pp. 16 18, Academic Press, New York Myers, D. K. and S|ater, E. C. (1957) Biochem. J. 67, 558-572 Cleland, K. W. and Slater, E. C. (1953) Biochem. J. 53, 547-556 Haugaard, N., Lee, H. H., Kostrzewa, R., Horn, R. S. and Haugaard, E. (1969) Biochim. Biophys. Acta 172, 198-204 Gamble, Jr, J. L. and Mazur, J. A. (1967) J. BioL Chem. 242, 67-72 Chappell, J. B. (1966) Biochem. J. 100, 43P Vignais, P. V., Duee, E. D., Vignais, P. M., and Huet, J. (1966) Biochirn. Biophys. Acta 118, 465-483 Santi, R., Bruni, A., Contessa, A. R. and Luciani, S. (1962) Boll. Soc. ltal. Biol. Sper. 38, 18901891