Comp. Biochem. Physiol. Vol. 10211,No. 3, pp. 635-638, 1992 Printed in Great Britain
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
REGULATION OF OXIDATIVE ACTIVITY A N D A ~ OF LIVER MITOCHONDRIA OF ACTIVE A N D HIBERNATING GOPHERS. THE ROLE OF PHOSPHOLIPASE A2 N. N. BRUSTOVETSKY,*M. V. EGOROVAand E. I. MAYEVSKY Institute of Theoretical and Experimental Biophysics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region, 142292, Russia (Received 11 October 1991)
Abstract--1. In the present work the initially lowered oxidase activity of liver mitochondria of hibernating gophers is shown to increase upon Ca2+-loading, after freezing-thawing repeated three times and after swelling in a medium containing potassium acetate as well as in a hypotonic sucrose medium. 2. In all cases the inhibition of phospholipase A2 hindered the increase of the oxidase activity of mitochondria. 3. Mitochondria of hibernating gophers have a lowered A~v in comparison with active animals, which is restored in the hypotonic medium.
INTRODUCTION Hibernation of mammals is a unique phenomenon in the living world. In this state the intensity of the oxidative metabolism of animals becomes 50-100 times lower and the body temperature falls to 4-5°C (Wang, 1987). The processes occurring in mitochondria, which are the main oxygen consumers and produce most heat in the cell, play an important role in the transition of animals from an active state to hibernation (Himms-Hagen, 1976). At present the liver mitochondria of hibernating animals are best studied. It has been established that the isolated liver mitochondria of hibernating animals have a considerably lower respiration rate in all metabolic states as compared to those of active ones (Fedotcheva et al., 1985; Pehowich and Wang, 1987; Gehnrich and Aprille, 1988; Brustovetsky et al., 1989; Brustovetsky et al., 1990). The suppression of respiration results from the inhibition of electron transfer in the bc~complex region of the respiratory chain (Gehnrich and Aprille, 1988; Brustovetsky et al., 1989; Brustovetsky et aL, 1990). The mechanism of the inhibition of electron transfer is as yet unknown. The inhibition of electron transfer in the respiratory chain is known to be accompanied by a decrease in A~u (Sorgato et al., 1985). So, it seems reasonable to assume that in the case of liver mitochondria from hibernating animals, whose respiratory chain is partly blocked, AM is also lowered. However, the measurements of AM in the liver mitochondria of hibernating gophers have not been carried out up to now. In our *To whom correspondence should be addressed. Abbreviations--A~P, potential on the inner mitochondrial membrane; TPP +, tetraphenylphosphonium; TMPD, N,N,N',N',-tetramethyl-n-phenylene diamine; DNP, 2,4-dinitrophenol; p-BPB, p-bromphenacylbromide; DCCD, N,N'-dicyclohexylcarbodiimide.
opinion, knowledge of AM value would allow us to explain easily the suppression of mitochondrial energy-dependent processes taking place during hibernation. The purpose of this work was mainly aimed at estimating A ~v in the liver mitochondria of active and hibernating gophers, as well as at studying the mechanism of the inhibition of electron transfer. For this purpose we made an attempt to remove the block of the respiratory chain by acting in various ways on the mitochondria of hibernating gophers. These experiments have demonstrated the possibility of removal of the block in the respiratory chain, activation of respiration and restoration of AM in the mitochondria of hibernating animals. Phospholipase A2 turned out to be of great significance for this process.
MATERIALS AND METHODS
In the experiments, active and hibernating gophers Citellus undulatus have been used. The body temperature of active gophers was about 37°C and that of hibernating ones about 5°C. The animals were kept in the conditions close to the natural ones. All the experiments were carried out in winter. Liver mitochondria were isolated by differential centrifugation in a medium containing 300 mM sucrose and 10 mM Tris-HCl, pH 7.4. Sucrose was first purified by using ion-exchange resin Dowex 50W× 8 (H+-form, Serva, Germany). Centrifugation was initially performed at 600 g for 10 min and then at 10,000g for 15 min. The mitochondria were kept on ice, the protein concentration in the suspension was 75-90mg/ml. The protein concentration was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard. The A~ value was estimated by K ÷ distribution between the medium and the mitochondrial matrix in the presence of valinomycin (Mitchell and Moyle, 1969) and by distribution of the lipophilic cation TPP + (Kamo et al., 1979; Lotscher et al., 1980). This procedure was based on the electron microscopy data according to which the isolated liver
635
636
N. N. BRUSTOVETSKYet al. Table 1. Respiration rate and A~' value of liver mitochondria of active and hibernating gophers. Effects of hypotonicity, antimycin A, myxothiazol and inhibitors of phospholipase A2 Groups of animals Active Active Active Hibern. Active Hibern. Hibern. Hibern.
Medium ton±city (mOsm)
Additions
250 250 250 250 60 60 60 60
-Antimycin A Myxothiaz ---p-BPB EGTA + A 23187
Respiration rate (nmoles O2/min/mg protein) + ADP - ADP + DNP 136 _ 7 (6) 40 + 3 (6) 44 ± 4 (4) 38 + 2 (6) 120+6(5) 83+5(5) 35+3(5) 38_+3(5)
30 + 3 (6) 21 _+2 (6) 25 + 3 (4) 27 ± 3 (6) 65+4(5) 40+3(5) 18+2(5) 20-+2(5)
AtP (mV)
148 + 6 (6) 46 + 4 (6) 48 ± 4 (4) 40 + 2 (6) 141_+9(5) 102+7(5) 39_+4(5) 42_+3(5)
196 4- 2 (8) 174 ± 4 (8) 176 ± 4 (5) 175 + 3 (10) 192+4(8) 189_+4(10) 171 _+3(10) 173_+2(10)
Substrate, 4 mM suceinate. Additions: 25/~M p-BPB; I mM EGTA; 0.2 #g/ml A 23187; 100 #M ADP; 40pM DNP; 5 10 SM antimycin A; 10-7M myxothiazol. Figures in parentheses indicate the number of experiments. The incubation medium and the conditions of the experiments are described in Materials and Methods. A~P was measured by K + distribution in the presence of valinomycin (Mitchell and Moyle, 1969). In all cases the mitochondria were incubated for not less than 5 min in the corresponding medium before ADP or DNP addition as well as before measuring A~. The data are presented as means + SD of the number of experiments indicated (N). mitochondria of active and hibernating gophers have an approximately equal volume (Fedotcheva et al., 1984). The volume of the mitochondrial matrix was taken as 1.0 #l/mg protein in the medium with normal ton±city (conditionally 250mOsm) containing 250mM sucrose, 3 mM MgCl2, 3 m M KH2PO4, 10mM Tris, pH7.4 and 1.5#l/mg of protein in a hypotonic medium (conditionally 60 mOsm) containing 6 0 m M sucrose and all the other components (Harris and Van Dam, 1968). The K ÷ concentration was measured by a K+-selective electrode; the TPP + concentration in the medium was measured by a TPP+-sensitive electrode. The respiration rate of mitochondria was measured polarographically using an oxygen Clark electrode in a thermostatic cell of 2.0 ml in volume by continuous stirring at 27°C in the media described above. In the experiments we used malate, glutamate, suecinate, ascorbate, TMPD, ADP, 2,4-dinitrophenol, Tris, bovine serum albumin, Lubrol WX, Triton X-100 from Serva (Germany), p-bromphenacylbromide, EGTA, valinomycin, A23187, N,N-dicyclohexylcarbodiimide from Sigma (St Louis, MO) and TPP ÷, myxothiazol from Fluka (Switzerland). RESULTS AND DISCUSSION In the e x p e r i m e n t s p e r f o r m e d the r e s p i r a t i o n rate in the liver m i t o c h o n d r i a f r o m h i b e r n a t i n g g o p h e r s was essentially lower, especially in the p h o s p h o r y l a t ing a n d u n c o u p l e d states, t h a n in the m i t o c h o n d r i a f r o m active a n i m a l s (Table 1). This w h o l l y coincides with t h e results o f o u r p r e v i o u s w o r k s ( B r u s t o v e t s k y et al., 1989; B r u s t o v e t s k y et al., 1990) a n d with the d a t a o b t a i n e d by o t h e r a u t h o r s as well ( F e d o t c h e v a et al., 1985; P e h o w i c h a n d W a n g , 1987; G e h n r i c h a n d Aprille, 1988). A s we s h o w e d earlier, t h e b r e a k i n g o f e l e c t r o n t r a n s f e r in the r e s p i r a t o r y c h a i n b c l - c o m p l e x r e g i o n was the cause o f the s u p p r e s s i o n o f o x i d a s e activities in the m i t o c h o n d r i a ( B r u s t o v e t s k y et al., 1990). O t h e r a u t h o r s have c o m e to similar c o n clusions ( G e h n r i c h a n d Aprille, 1988). A s a rule, t h e i n h i b i t i o n o f the r e s p i r a t o r y c h a i n results in a decrease o f A~u ( S o r g a t o et al., 1985). T h e r e f o r e , there were m a n y r e a s o n s to suggest t h a t the liver m i t o c h o n dria f r o m h i b e r n a t i n g g o p h e r s have a l o w e r e d A ~ in c o m p a r i s o n w i t h t h o s e f r o m active animals. Similar results were o b t a i n e d in the d e t e r m i n a t i o n o f A ~ by K + d i s t r i b u t i o n in the p r e s e n c e o f v a l i n o m y c i n a n d by d i s t r i b u t i o n o f t h e lipophilic c a t i o n T P P +
(Tables 1 a n d 2; Fig. 1). It is n o t e w o r t h y t h a t the differences in the A ~ m a g n i t u d e were o b s e r v e d w h e n g l u t a m a t e plus m a l a t e o r succinate were used as s u b s t r a t e s (Table 2). In the case o f the o x i d a t i o n o f a s c o r b a t e plus T M P D t h e A ~o values were practically the s a m e for t h e m i t o c h o n d r i a f r o m b o t h g r o u p s o f animals. W h e n g l u t a m a t e plus m a l a t e or succinate were oxidized, distinct differences in the sensitivity o f A ~ to t h e a c t i o n o f low c o n c e n t r a t i o n s o f the u n c o u p l e r D N P were o b s e r v e d b e t w e e n the liver m i t o c h o n d r i a o f active a n d h i b e r n a t i n g animals. T h e a d d i t i o n o f D N P led to a m o r e c o n s i d e r a b l e fall o f A ~ in t h e liver m i t o c h o n d r i a o f h i b e r n a t i n g g o p h e r s as c o m p a r e d to t h o s e o f active ones (Fig. 1). T h e i n h i b i t i o n o f r e s p i r a t o r y c h a i n and, as a result, a decrease in the intensity o f the f u n c t i o n i n g o f the p r o t o n p u m p s , are o b v i o u s l y the c h i e f r e a s o n s for t h e h i g h e r sensitivity o f A ~ in t h e m i t o c h o n d r i a f r o m h i b e r n a t i n g g o p h e r s to the u n c o u p l e r action. Partial i n h i b i t i o n o f the r e s p i r a t o r y c h a i n in the liver m i t o c h o n d r i a f r o m active a n i m a l s by a n t i m y c i n A o r m y x o t h i a z o l resulting in the s u p p r e s s i o n o f r e s p i r a t i o n to a level typical for the m i t o c h o n d r i a f r o m h i b e r n a t i n g g o p h e r s c a u s e d a d e c r e a s e o f A~u (Table 1) a n d a c o n s i d e r a b l e increase o f its sensitivity to the unc o u p l e r a c t i o n (Fig. 1). T h e a d d i t i o n o f A D P to the
Table 2. A~ value of the liver mitochondria of active and hibernating gophers oxidizing the different substrates in the medium with various ton±city
Groups of animals
Medium ton±city Malate, (mOsm) glutamate
A~' (mV) Substrates: Succinate
Ascorbate, TMPD
Active 250 185 + 3 196 _+2 200 ± 4 Active 60 188 ± 4 192 + 4 198 ± 3 Hibern. 250 164 + 3 175 + 3 197 + 4 Hibern. 60 183 + 2 189 + 4 199 ± 5 Substrates: 2.5 mM malate, 4 mM glutamate, 4 mM succinate, 4 mM ascorbate, 300/~M TMPD. A~ was measured by K + distribution in the presence of valinomycin (Mitchell and Moyle, 1969). In all cases the mitochondria were incubated for not less than 5 min in the corresponding medium before measuring A~. Incubation mediums are described in Materials and Methods. In all cases N = 8-10. The data are presented as means + SD of the number of experiments indicated (N).
Mitochondrial oxidation in gophers
18°I
l'~jmV 200F
DNP
637
•f,mY 20C.
ADP ~
180
/ / / ' ~ ~
Olig°mycin DCCD or
140
2 min 120 L
, 2 min
Fig. 1. A~ values in the liver mitochondria from active (1,2) and hibernating gophers measured by a TPP÷-sensitive electrode. Effects of 2,4-dinitrophenol, antimycin A, myxothiazol and hypotonicity. (I) Mitochondria of an active gopher (normal medium). (2) Mitochondria of an active gopher in the presence of 5 10-s M antimycin A or 10 -7 M myxothiazol (normal medium). (3) Mitochondria of a hibernating gopher (hypotonic medium). (4) Mitochondria of a hibernating gopher (normal medium). Substrate, 4 mM succinate. Additions: DNP---cach arrow points to 5 pM. The incubation medium is described in Materials and Methods.
liver mitochondria from active animals resulted in a fall of A ~ followed by its rapid restoration (Fig. 2). Similar changes in A~v were observed when A D P was added to the liver mitochondria of rats. The A D P addition to the liver mitochondria from hibernating gophers also caused a decrease in A~v. However, unlike the mitochondria of active gophers or rats, the A ~ restoration stage slowed down greatly (Fig. 2). The addition of oligomycin or D C C D to the mitochondria from hibernating animals after A D P addition led to a rapid restoration of A ~ (Fig. 2). Obviously, the A D P addition to the liver mitochondria of hibernating gophers induces a leakage of H ÷ through the proton channel of ATP-synthetase, which is not compensated for by the proton pumps of the respiratory chain. This phenomenon is not specific to the mitochondria of hibernating gophers and is easily reproduced upon a slight A ~ decrease as a result of the partial inhibition of the respiratory chain in the liver mitochondria of active gophers or rats by antimycin A or myxothiazol.
120 Fig. 2. Effects of ADP, oligomycin and DCCD on A~ of liver mitochondria of active and hibernating gophers. (1) Mitochondria of an active gopher (normal medium). (2) Mitochondria of a hibernating gopher (normal medium). (3) Mitochondria of a hibernating gopher (hypotonic medium). Substrate, 4 mM succinate. Additions: 100 #M ADP; 2/~ M oligomycin (Oligo); 25 #M DCCD. The dotted line shows A~' in the absence of oligomycin or DCCD. The decrease in the tonicity of the incubation medium led to a substantial activation of mitochondrial respiration in hibernating gophers practically without any effect on the respiration of mitochondria in active animals. In this case there was only an increase of the respiration rate in the 4th metabolic state (Table 1). The enhancement of the oxidase activities of liver mitochondria in hibernating animals also took place upon their loading with Ca 2+, their swelling in an isotonic medium containing potassium acetate (especially in the presence of valinomycin), and after the freezing-thawing procedure, repeated three times in a sucrose medium with normal tonicity (Table 3). The detergent Lubrol W X (or Triton X-100), which also leads to a weakening of the barrier properties of the inner mitochondrial membrane, did not cause an enhancement of the oxidase activities of liver mitochondria from hibernating gophers. The increase of A ~ in the liver mitochondria of hibernating gophers was only observed in the conditions of hypotonicity (Table 2; Fig. 1). At the same time the A~g sensitivity to the uncoupler decreased (Fig. 1); the dynamics of the A~u change in response to A D P addition approached that of the A ~ change in active animals
Table 3. Succinate-oxidase activity of the hibernating gopher liver mitochondria under different actions Rate of respiration (ng at O2/min-I/mg protein) Experimental Additions: actions Control Experiment +p-BPB + EGTA, A 23187 Ca2+-load 40_+4 (7) 110_+8(7) 42_+3 (5) -Freezing-thawing 36 _+3 (6) 85 _+9 (6) 44 _+5 (5) 42 + 4 (5) K÷-acetate medium 37 _+4 (5) 89 _+7 (5) 39 _+2 (5) 45 _+3 (4) Lubrol WX* 44.5 (4) 39 _+4 (6) 40 _+4 (5) 42 _+5 (5) Substrate: 4 mM succinate. Additions: 75 nmoles Ca 2+ per mg protein; 0.1% Lubrol WX; 25 #M p-BPB; 1 mM EGTA; 0.2#g/ml A 23187. All experiments were performed in the presence of 40 # M DNP. Figures in parentheses indicate the number of experiments. *Similar results were obtained with Triton X-100 (0.1%). The data are presented as means -+SD of the number of experiments indicated (N).
638
N. N. BRUSTOVETSKYel al.
(Fig. 2). There were practically no changes in the A ~ magnitude of liver mitochondria of active gophers in the hypotonic medium. The inhibition of the mitochondrial phospholipase A 2 by p-bromphenacylbromide (De Winter et al., 1984; Chang et al., 1987) or the inhibition resulting from the considerable exhaustion of the mitochondrial Ca 2+ pool by E G T A plus A23187 prevented the enhancement of the oxidase activities of liver mitochondria in hibernating gophers in all cases and the A ~ increase under conditions of hypotonicity (Table 1). The inhibitors of phospholipase A 2 did not influence the respiration of liver mitochondria of active gophers. As a matter of fact, the inhibition of phospholipase A2 did not affect the swelling of mitochondria in the hypotonic medium, either. Thus, it can be concluded that the activation of phospholipase A 2 in hypotonicity (Kargapolov, 1979), upon freezing-thawing (Waite et al., 1969) and Ca2+-loading (Nachbaur et aL, 1972) promotes the removal of the block in the respiratory chain and is very significant for the regulation of oxidase activities and the A7j magnitude (in the case of hypotonicity) in the liver mitochondria of hibernating gophers. REFERENCES
Brustovetsky N. N., Amerkhanov Z. G., Popova E. Yu. and Konstantinov A. A. (1990) Reversible inhibition of electron transfer in the ubiquinol cytochrome c reductase segment of the mitochondrial respiratory chain in hibernating ground squirrels. FEBS Lett. 263, 73~6. Brustovetsky N. N., Mayevsky E. I., Grishina E. V., Gogvadze V. G. and Amerkhanov Z. G. (1989) Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus. Comp. Biochem. Physiol. 94B, 537-541. Chang J., Musser J. H. and McGregor H. (1987) Phospholipase A2: function and pharmacological regulation. Biochem. Pharmac. 36, 2429-2436. De Winter J. M., Korpancova J. and Van den Bosch H. (1984) Regulatory aspects of rat liver mitochondrial phospholipase A2: effects of calcium ions and calmodulin. Archs Biochem. Biophys. 234, 243-252. Fedotcheva N. J., Mironova G. D., lljasova E. N. and Kolaeva S. G. (1984) Appearance of the external pathway of NADH oxidation in mitochondria during hibernation. Cryo-Lett. 5, 27-32. Fedotcheva N. J., Sharyshev A. A., Mironova G. D. and Kondrashova M. N. (1985) Inhibition of succinate
oxidation and K + transport in mitochondria during hibernation. Comp. Biochem. Physiol. 82B, 191-195. Gehnrich S. C. and Aprille J. R. (1988) Hepatic gluconeogenesis and mitochondrial function during hibernation. Comp. Biochem. Physiol. 91B, 11-16. Harris E. J. and Van Dam K. (1968) Changes of total water and sucrose space accompanying induced ion uptake or phosphate swelling of rat liver mitochondria. Biochem. J. 106, 759-766. Himms-Hagen J. (1976) Cellular thermogenesis. A. Rev. Physiol. 38, 315-351. Kamo N., Muratsugu M., Hongoh R. and Kobatake Y. (1979) Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and relationship between proton electrochemical potential and phosphorylation potential in steady-state. J. Membr. Biol. 49, 105 121. Kargapolov A. V. (1979) Changes in the phospholipid content of intact mitochondria under mitochondrial swelling in hypotonic sucrose solutions. Biochimica 44, 293-296 (in Russian). Lotscher H.-R., Winterhalter K. H., Carafoli E. and Richter C. (1980) The energy-state of mitochondria during the transport of Ca 2÷. Eur. J. Biochem. 110, 21 l-B16. Lowry O. 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. Mitchell P. and Moyle J. (1969) Estimation of membrane potential and pH difference across the cristae membrane of rat liver mitochondria. Eur. J. Biochem. 7, 471-484. Nachbaur J., Colbeau A. and Vignais P. M. (1972) Distribution of membrane-confined phospholipases A in the rat hepatocyte. Biochim. biophys. Acta 274, 426-446. Wang L. C. H. (1987) Mammalian hibernation. In The Effect of Low Temperatures on Biological Systems (Edited by Grout B. W. W. and Morris G. J.), pp. 349-385. Edward Arnold, London. Pehowich D. J. and Wang L. C. H. (1987) Stoichiometry of H + etttux to respiration-dependent Ca 2+ uptake and oxygen consumption in liver mitochondria from a hibernator. Physiol. Zool. 60, 114-120. Sorgato M. C., Lippe, Seren S. and Ferguson S. J. (1985) Partial uncoupling, or inhibition of electron transport rate, have equivalent effects on the relationship between the rate of ATP synthesis and proton-motive force in submitochondrial particles. FEBS Lett. 181, 323-327. Waite M., Scherphof G. L., Boshouwerst F. M. G. and Van Deenen L. L. M. (1969) Differentiation of phospholipase A in mitochondria and lysosomes of rat liver. J. Lipid Res. 10, 411-420.
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
REGULATION OF OXIDATIVE ACTIVITY A N D A ~ OF LIVER MITOCHONDRIA OF ACTIVE A N D HIBERNATING GOPHERS. THE ROLE OF PHOSPHOLIPASE A2 N. N. BRUSTOVETSKY,*M. V. EGOROVAand E. I. MAYEVSKY Institute of Theoretical and Experimental Biophysics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region, 142292, Russia (Received 11 October 1991)
Abstract--1. In the present work the initially lowered oxidase activity of liver mitochondria of hibernating gophers is shown to increase upon Ca2+-loading, after freezing-thawing repeated three times and after swelling in a medium containing potassium acetate as well as in a hypotonic sucrose medium. 2. In all cases the inhibition of phospholipase A2 hindered the increase of the oxidase activity of mitochondria. 3. Mitochondria of hibernating gophers have a lowered A~v in comparison with active animals, which is restored in the hypotonic medium.
INTRODUCTION Hibernation of mammals is a unique phenomenon in the living world. In this state the intensity of the oxidative metabolism of animals becomes 50-100 times lower and the body temperature falls to 4-5°C (Wang, 1987). The processes occurring in mitochondria, which are the main oxygen consumers and produce most heat in the cell, play an important role in the transition of animals from an active state to hibernation (Himms-Hagen, 1976). At present the liver mitochondria of hibernating animals are best studied. It has been established that the isolated liver mitochondria of hibernating animals have a considerably lower respiration rate in all metabolic states as compared to those of active ones (Fedotcheva et al., 1985; Pehowich and Wang, 1987; Gehnrich and Aprille, 1988; Brustovetsky et al., 1989; Brustovetsky et al., 1990). The suppression of respiration results from the inhibition of electron transfer in the bc~complex region of the respiratory chain (Gehnrich and Aprille, 1988; Brustovetsky et al., 1989; Brustovetsky et aL, 1990). The mechanism of the inhibition of electron transfer is as yet unknown. The inhibition of electron transfer in the respiratory chain is known to be accompanied by a decrease in A~u (Sorgato et al., 1985). So, it seems reasonable to assume that in the case of liver mitochondria from hibernating animals, whose respiratory chain is partly blocked, AM is also lowered. However, the measurements of AM in the liver mitochondria of hibernating gophers have not been carried out up to now. In our *To whom correspondence should be addressed. Abbreviations--A~P, potential on the inner mitochondrial membrane; TPP +, tetraphenylphosphonium; TMPD, N,N,N',N',-tetramethyl-n-phenylene diamine; DNP, 2,4-dinitrophenol; p-BPB, p-bromphenacylbromide; DCCD, N,N'-dicyclohexylcarbodiimide.
opinion, knowledge of AM value would allow us to explain easily the suppression of mitochondrial energy-dependent processes taking place during hibernation. The purpose of this work was mainly aimed at estimating A ~v in the liver mitochondria of active and hibernating gophers, as well as at studying the mechanism of the inhibition of electron transfer. For this purpose we made an attempt to remove the block of the respiratory chain by acting in various ways on the mitochondria of hibernating gophers. These experiments have demonstrated the possibility of removal of the block in the respiratory chain, activation of respiration and restoration of AM in the mitochondria of hibernating animals. Phospholipase A2 turned out to be of great significance for this process.
MATERIALS AND METHODS
In the experiments, active and hibernating gophers Citellus undulatus have been used. The body temperature of active gophers was about 37°C and that of hibernating ones about 5°C. The animals were kept in the conditions close to the natural ones. All the experiments were carried out in winter. Liver mitochondria were isolated by differential centrifugation in a medium containing 300 mM sucrose and 10 mM Tris-HCl, pH 7.4. Sucrose was first purified by using ion-exchange resin Dowex 50W× 8 (H+-form, Serva, Germany). Centrifugation was initially performed at 600 g for 10 min and then at 10,000g for 15 min. The mitochondria were kept on ice, the protein concentration in the suspension was 75-90mg/ml. The protein concentration was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard. The A~ value was estimated by K ÷ distribution between the medium and the mitochondrial matrix in the presence of valinomycin (Mitchell and Moyle, 1969) and by distribution of the lipophilic cation TPP + (Kamo et al., 1979; Lotscher et al., 1980). This procedure was based on the electron microscopy data according to which the isolated liver
635
636
N. N. BRUSTOVETSKYet al. Table 1. Respiration rate and A~' value of liver mitochondria of active and hibernating gophers. Effects of hypotonicity, antimycin A, myxothiazol and inhibitors of phospholipase A2 Groups of animals Active Active Active Hibern. Active Hibern. Hibern. Hibern.
Medium ton±city (mOsm)
Additions
250 250 250 250 60 60 60 60
-Antimycin A Myxothiaz ---p-BPB EGTA + A 23187
Respiration rate (nmoles O2/min/mg protein) + ADP - ADP + DNP 136 _ 7 (6) 40 + 3 (6) 44 ± 4 (4) 38 + 2 (6) 120+6(5) 83+5(5) 35+3(5) 38_+3(5)
30 + 3 (6) 21 _+2 (6) 25 + 3 (4) 27 ± 3 (6) 65+4(5) 40+3(5) 18+2(5) 20-+2(5)
AtP (mV)
148 + 6 (6) 46 + 4 (6) 48 ± 4 (4) 40 + 2 (6) 141_+9(5) 102+7(5) 39_+4(5) 42_+3(5)
196 4- 2 (8) 174 ± 4 (8) 176 ± 4 (5) 175 + 3 (10) 192+4(8) 189_+4(10) 171 _+3(10) 173_+2(10)
Substrate, 4 mM suceinate. Additions: 25/~M p-BPB; I mM EGTA; 0.2 #g/ml A 23187; 100 #M ADP; 40pM DNP; 5 10 SM antimycin A; 10-7M myxothiazol. Figures in parentheses indicate the number of experiments. The incubation medium and the conditions of the experiments are described in Materials and Methods. A~P was measured by K + distribution in the presence of valinomycin (Mitchell and Moyle, 1969). In all cases the mitochondria were incubated for not less than 5 min in the corresponding medium before ADP or DNP addition as well as before measuring A~. The data are presented as means + SD of the number of experiments indicated (N). mitochondria of active and hibernating gophers have an approximately equal volume (Fedotcheva et al., 1984). The volume of the mitochondrial matrix was taken as 1.0 #l/mg protein in the medium with normal ton±city (conditionally 250mOsm) containing 250mM sucrose, 3 mM MgCl2, 3 m M KH2PO4, 10mM Tris, pH7.4 and 1.5#l/mg of protein in a hypotonic medium (conditionally 60 mOsm) containing 6 0 m M sucrose and all the other components (Harris and Van Dam, 1968). The K ÷ concentration was measured by a K+-selective electrode; the TPP + concentration in the medium was measured by a TPP+-sensitive electrode. The respiration rate of mitochondria was measured polarographically using an oxygen Clark electrode in a thermostatic cell of 2.0 ml in volume by continuous stirring at 27°C in the media described above. In the experiments we used malate, glutamate, suecinate, ascorbate, TMPD, ADP, 2,4-dinitrophenol, Tris, bovine serum albumin, Lubrol WX, Triton X-100 from Serva (Germany), p-bromphenacylbromide, EGTA, valinomycin, A23187, N,N-dicyclohexylcarbodiimide from Sigma (St Louis, MO) and TPP ÷, myxothiazol from Fluka (Switzerland). RESULTS AND DISCUSSION In the e x p e r i m e n t s p e r f o r m e d the r e s p i r a t i o n rate in the liver m i t o c h o n d r i a f r o m h i b e r n a t i n g g o p h e r s was essentially lower, especially in the p h o s p h o r y l a t ing a n d u n c o u p l e d states, t h a n in the m i t o c h o n d r i a f r o m active a n i m a l s (Table 1). This w h o l l y coincides with t h e results o f o u r p r e v i o u s w o r k s ( B r u s t o v e t s k y et al., 1989; B r u s t o v e t s k y et al., 1990) a n d with the d a t a o b t a i n e d by o t h e r a u t h o r s as well ( F e d o t c h e v a et al., 1985; P e h o w i c h a n d W a n g , 1987; G e h n r i c h a n d Aprille, 1988). A s we s h o w e d earlier, t h e b r e a k i n g o f e l e c t r o n t r a n s f e r in the r e s p i r a t o r y c h a i n b c l - c o m p l e x r e g i o n was the cause o f the s u p p r e s s i o n o f o x i d a s e activities in the m i t o c h o n d r i a ( B r u s t o v e t s k y et al., 1990). O t h e r a u t h o r s have c o m e to similar c o n clusions ( G e h n r i c h a n d Aprille, 1988). A s a rule, t h e i n h i b i t i o n o f the r e s p i r a t o r y c h a i n results in a decrease o f A~u ( S o r g a t o et al., 1985). T h e r e f o r e , there were m a n y r e a s o n s to suggest t h a t the liver m i t o c h o n dria f r o m h i b e r n a t i n g g o p h e r s have a l o w e r e d A ~ in c o m p a r i s o n w i t h t h o s e f r o m active animals. Similar results were o b t a i n e d in the d e t e r m i n a t i o n o f A ~ by K + d i s t r i b u t i o n in the p r e s e n c e o f v a l i n o m y c i n a n d by d i s t r i b u t i o n o f t h e lipophilic c a t i o n T P P +
(Tables 1 a n d 2; Fig. 1). It is n o t e w o r t h y t h a t the differences in the A ~ m a g n i t u d e were o b s e r v e d w h e n g l u t a m a t e plus m a l a t e o r succinate were used as s u b s t r a t e s (Table 2). In the case o f the o x i d a t i o n o f a s c o r b a t e plus T M P D t h e A ~o values were practically the s a m e for t h e m i t o c h o n d r i a f r o m b o t h g r o u p s o f animals. W h e n g l u t a m a t e plus m a l a t e or succinate were oxidized, distinct differences in the sensitivity o f A ~ to t h e a c t i o n o f low c o n c e n t r a t i o n s o f the u n c o u p l e r D N P were o b s e r v e d b e t w e e n the liver m i t o c h o n d r i a o f active a n d h i b e r n a t i n g animals. T h e a d d i t i o n o f D N P led to a m o r e c o n s i d e r a b l e fall o f A ~ in t h e liver m i t o c h o n d r i a o f h i b e r n a t i n g g o p h e r s as c o m p a r e d to t h o s e o f active ones (Fig. 1). T h e i n h i b i t i o n o f r e s p i r a t o r y c h a i n and, as a result, a decrease in the intensity o f the f u n c t i o n i n g o f the p r o t o n p u m p s , are o b v i o u s l y the c h i e f r e a s o n s for t h e h i g h e r sensitivity o f A ~ in t h e m i t o c h o n d r i a f r o m h i b e r n a t i n g g o p h e r s to the u n c o u p l e r action. Partial i n h i b i t i o n o f the r e s p i r a t o r y c h a i n in the liver m i t o c h o n d r i a f r o m active a n i m a l s by a n t i m y c i n A o r m y x o t h i a z o l resulting in the s u p p r e s s i o n o f r e s p i r a t i o n to a level typical for the m i t o c h o n d r i a f r o m h i b e r n a t i n g g o p h e r s c a u s e d a d e c r e a s e o f A~u (Table 1) a n d a c o n s i d e r a b l e increase o f its sensitivity to the unc o u p l e r a c t i o n (Fig. 1). T h e a d d i t i o n o f A D P to the
Table 2. A~ value of the liver mitochondria of active and hibernating gophers oxidizing the different substrates in the medium with various ton±city
Groups of animals
Medium ton±city Malate, (mOsm) glutamate
A~' (mV) Substrates: Succinate
Ascorbate, TMPD
Active 250 185 + 3 196 _+2 200 ± 4 Active 60 188 ± 4 192 + 4 198 ± 3 Hibern. 250 164 + 3 175 + 3 197 + 4 Hibern. 60 183 + 2 189 + 4 199 ± 5 Substrates: 2.5 mM malate, 4 mM glutamate, 4 mM succinate, 4 mM ascorbate, 300/~M TMPD. A~ was measured by K + distribution in the presence of valinomycin (Mitchell and Moyle, 1969). In all cases the mitochondria were incubated for not less than 5 min in the corresponding medium before measuring A~. Incubation mediums are described in Materials and Methods. In all cases N = 8-10. The data are presented as means + SD of the number of experiments indicated (N).
Mitochondrial oxidation in gophers
18°I
l'~jmV 200F
DNP
637
•f,mY 20C.
ADP ~
180
/ / / ' ~ ~
Olig°mycin DCCD or
140
2 min 120 L
, 2 min
Fig. 1. A~ values in the liver mitochondria from active (1,2) and hibernating gophers measured by a TPP÷-sensitive electrode. Effects of 2,4-dinitrophenol, antimycin A, myxothiazol and hypotonicity. (I) Mitochondria of an active gopher (normal medium). (2) Mitochondria of an active gopher in the presence of 5 10-s M antimycin A or 10 -7 M myxothiazol (normal medium). (3) Mitochondria of a hibernating gopher (hypotonic medium). (4) Mitochondria of a hibernating gopher (normal medium). Substrate, 4 mM succinate. Additions: DNP---cach arrow points to 5 pM. The incubation medium is described in Materials and Methods.
liver mitochondria from active animals resulted in a fall of A ~ followed by its rapid restoration (Fig. 2). Similar changes in A~v were observed when A D P was added to the liver mitochondria of rats. The A D P addition to the liver mitochondria from hibernating gophers also caused a decrease in A~v. However, unlike the mitochondria of active gophers or rats, the A ~ restoration stage slowed down greatly (Fig. 2). The addition of oligomycin or D C C D to the mitochondria from hibernating animals after A D P addition led to a rapid restoration of A ~ (Fig. 2). Obviously, the A D P addition to the liver mitochondria of hibernating gophers induces a leakage of H ÷ through the proton channel of ATP-synthetase, which is not compensated for by the proton pumps of the respiratory chain. This phenomenon is not specific to the mitochondria of hibernating gophers and is easily reproduced upon a slight A ~ decrease as a result of the partial inhibition of the respiratory chain in the liver mitochondria of active gophers or rats by antimycin A or myxothiazol.
120 Fig. 2. Effects of ADP, oligomycin and DCCD on A~ of liver mitochondria of active and hibernating gophers. (1) Mitochondria of an active gopher (normal medium). (2) Mitochondria of a hibernating gopher (normal medium). (3) Mitochondria of a hibernating gopher (hypotonic medium). Substrate, 4 mM succinate. Additions: 100 #M ADP; 2/~ M oligomycin (Oligo); 25 #M DCCD. The dotted line shows A~' in the absence of oligomycin or DCCD. The decrease in the tonicity of the incubation medium led to a substantial activation of mitochondrial respiration in hibernating gophers practically without any effect on the respiration of mitochondria in active animals. In this case there was only an increase of the respiration rate in the 4th metabolic state (Table 1). The enhancement of the oxidase activities of liver mitochondria in hibernating animals also took place upon their loading with Ca 2+, their swelling in an isotonic medium containing potassium acetate (especially in the presence of valinomycin), and after the freezing-thawing procedure, repeated three times in a sucrose medium with normal tonicity (Table 3). The detergent Lubrol W X (or Triton X-100), which also leads to a weakening of the barrier properties of the inner mitochondrial membrane, did not cause an enhancement of the oxidase activities of liver mitochondria from hibernating gophers. The increase of A ~ in the liver mitochondria of hibernating gophers was only observed in the conditions of hypotonicity (Table 2; Fig. 1). At the same time the A~g sensitivity to the uncoupler decreased (Fig. 1); the dynamics of the A~u change in response to A D P addition approached that of the A ~ change in active animals
Table 3. Succinate-oxidase activity of the hibernating gopher liver mitochondria under different actions Rate of respiration (ng at O2/min-I/mg protein) Experimental Additions: actions Control Experiment +p-BPB + EGTA, A 23187 Ca2+-load 40_+4 (7) 110_+8(7) 42_+3 (5) -Freezing-thawing 36 _+3 (6) 85 _+9 (6) 44 _+5 (5) 42 + 4 (5) K÷-acetate medium 37 _+4 (5) 89 _+7 (5) 39 _+2 (5) 45 _+3 (4) Lubrol WX* 44.5 (4) 39 _+4 (6) 40 _+4 (5) 42 _+5 (5) Substrate: 4 mM succinate. Additions: 75 nmoles Ca 2+ per mg protein; 0.1% Lubrol WX; 25 #M p-BPB; 1 mM EGTA; 0.2#g/ml A 23187. All experiments were performed in the presence of 40 # M DNP. Figures in parentheses indicate the number of experiments. *Similar results were obtained with Triton X-100 (0.1%). The data are presented as means -+SD of the number of experiments indicated (N).
638
N. N. BRUSTOVETSKYel al.
(Fig. 2). There were practically no changes in the A ~ magnitude of liver mitochondria of active gophers in the hypotonic medium. The inhibition of the mitochondrial phospholipase A 2 by p-bromphenacylbromide (De Winter et al., 1984; Chang et al., 1987) or the inhibition resulting from the considerable exhaustion of the mitochondrial Ca 2+ pool by E G T A plus A23187 prevented the enhancement of the oxidase activities of liver mitochondria in hibernating gophers in all cases and the A ~ increase under conditions of hypotonicity (Table 1). The inhibitors of phospholipase A 2 did not influence the respiration of liver mitochondria of active gophers. As a matter of fact, the inhibition of phospholipase A2 did not affect the swelling of mitochondria in the hypotonic medium, either. Thus, it can be concluded that the activation of phospholipase A 2 in hypotonicity (Kargapolov, 1979), upon freezing-thawing (Waite et al., 1969) and Ca2+-loading (Nachbaur et aL, 1972) promotes the removal of the block in the respiratory chain and is very significant for the regulation of oxidase activities and the A7j magnitude (in the case of hypotonicity) in the liver mitochondria of hibernating gophers. REFERENCES
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