- Email: [email protected]
Butyric acid as a supressor of mitochondrial functions in Physarum polycephalum plasmodia
Cell Biology
International
Reports,
Vol. 11, No. 4, April
301
1987
BUTYRIC ACID AS A SUPRESSOROF MITOCHONDRIAL FUNCTIONS IN PHYSARUM POLYCEPHALUMPLASMODIA M Cieslawska, Nencki
Institute
B Hrebenda
of Experimental Biology, Warsaw, Poland
and Z Baranowski 3 Pasteur
Str.,
02-093
Abstract By means of tensionmetric technique, in the presence of butyric and acid, a comparison is made between the action of respiratory glycolytic inhibitors on plasmodial strands of Physarum polycephalum. It is shown that, in presence of this weak acid, glycolysis but not exidative phosphorylation can preserve the contractile ability of the samples. Inhibitory analysis has revealed that the inhibition of mitochondrial functions by butyric acid is non-specific in the sense that the drug influences both KCN-resistant In all probability the observed and KCN-sensitive respiration. responses of plasmodial strands to butyric acid are related to acidification of the cytoplasm. Introduction It is well established that intracellular pH of a variety of cell the steady-state of proton types is held near pH 7 and represents concentration. In Physarum polycephalum plasmodia internal pH [pHi] undergoes short term oscillations with average value of 6.6 + 0.5 and an amplitude of about 0.1 pH unit [Nakamura and The period of these oscillations coincides with a Kamiya, 19851. minute rhythm of the contraction-relaxation cycle of plasmodial actomyosin. In spite of long term changes of pHi related to mitotic cycle are observed [Gerson and Burton, 1977; Morisawa and Stainhard, 19821. In short term oscillations the phase of acidification coincides with the contraction phase of the contractionrelaxation cycle [Nakamura and Kamiya, 19851. However, the relation between the average value of pHi and contractility or chemotactic behaviour of plasmodia is not yet fully elucidated [Hirose et al., 1982; Matveeva et al., 1979, IgSl]. In the previous report [Cieslawska et al., 19861 we have observed an increase in the frequency of the tensiometrically measured contractile activity of plasmodial strands in the presence of a weak base [procaine] and a decrease in the frequency in response to externally applied weak acid [butyric acid]. These drugs penetrate into the cell in undissociated form and either raise [procaine] or lower [butyric acid] manner [Sanders and Slayman, 1982]. PHi in a concentration-dependent Recent findings concerning the regulation of the contractionrelaxation cycle of plasmodial contractile system [Korohoda et al., 1983; Baranowski, 1985b] allow one to carry out an analysis of the inhibitory influence of any drug on energy-yielding metabolic processes. The results presented in this paper show that the 0309-1651/871040301-06/$03.00/O
0 1987 Academic
Press Inc. (London)
Ltd.
302
Cell Biology
International
Reports,
Vol. 11, No. 4, April
1987
contractile response of plasmodial strand to butyric acid laefLects not cnly the generally accepted effect of decreaeing pH. but also c.auses the inhibition of mitochondrial functions in plaimodia. Material
and Methods
Plasmodial strands isolated from surface cultures of Physarum polycephalum were used for this study. The contractile activity of the strandstis been studied by means of a tension transducer under isometric conditions of measurements [Wohlfarth-Bottermann, 19753. The samples were submerged in salt solution containing: 2mM NaCl, 1 mM KCl, 7 mM MgC12, 1 mM CaC12; pH 5.0-5.2, at a constant temperature of 20°C2 maintained by the use of a Peltier element. The following concentrations of chemicals dissolved in the salt solution were used: 5 mM KCN, ImM iodoacetate [MIA] 10 mM ketoglutaric acid [KG], 10 mM pyruvic acid [pyr], 10 mM L-malic acid [mall, 7 mM salicylhydroxamic acid [SHAM], 10 mM butyric acid [but]. The chemicals were applied externally by replacing solutions surrounding the samples in the perfusion chamber. Results
and Discussion
As was mentioned in the Introduction, butyric acid applied externally lowers the frequency of force oscillations generated by plasmodial strands. To observe this effect [see Fig.11 two preconditions are indispensable: i - the period of oscillations before treatment must be relatively high [2-T min.] and ii - the pH of a solution has to be adjusted near to the value of the pK of butyric This induces a reduction in the internal pH [pHi] of acid [4.8]. The first precondition is the samples [Sanders and Slayman, 19821. The period of oscillthe same as in the case of KCN application. ations in the presence of KCN or butyric acid is about 4 min. inIt suggests that the dependent of its value before treatment. action of butyric acid is not restricted to decreasing pH. but also has an influence on energy metabolism of the plasmodial skrands. The aim of the experiments described in this paper was to check this hvDothesis.
Fig.
decrease of 1. Reversible frequency of force oscillations in response to 10 mM butyric acid [but].
Cell Biology
International
Reports,
Vol. 11, No. 4, April
1987
303
The lack of a reasonable hypothesis concerning processes responsible for oscillatory contraction activity of plasmodial actomyosin does not allow one to interpret unequivically the changes in the period However, the persistence of oscior amplitude of the oscillations. llations or the reversibility of their cessation in response to experimental procedure gives some information about disturbances in This kind of inhibitory analysis is energy-yielding processes. based on following observations previously published [Korohoda et al., 1983; Baranowski, 1985 a,b] [notes a - f]: a
plasmodial mitochondria exhibit pathway, which is KCN-resistant
b
inhibitors of glycolysis [eg. MIA], cytochrome oxidase [KCN], or alternative respiration [eg. SHAM] applied separately, over a time span of a few hours do not show any influence on the persistence of oscillations;
C
an alternative respiratory but SHAM-sensitive;
simultaneous inhibition of glycolysis oxidase [KCN] leads to an irreversible oscillations;
[MIA] and cytochrome cessation of the
d
alternative respiration is able to maintain the oscillations in the presence of KCN with MIA provided that it is supported by externally applied ketoglutarate [KG] or pyruvate with malate [PY~ mall;
e
the oscillations solely maintained by ketoglutarate or pyruvate with malate [ie. in the presence of KCN with MIA] irreversibly disappear after inhibition of the alternative respiratory pathway [SHAM];
f
total inhibition of respiration but not glycolysis [KCN with SHAM treatment] stops the oscillations in a reversible manner although the contractile ability is preserved. Fig. 2 Irreversible cessation of oscillations in response to butyric acid in the presence of iodoacetate [MIA]. FOG concentrations of chemicals and their abbreviations see Material and Methods.
304
Cell Biology
International
Reports,
Vol. 17, No. 4, April
7987
The tensiogram in Fig.2 shows that MIA [an inhibitor of glyceraldehyde-phosphate dehydrogenase] together with butyric acid causes the death of the plasmodial strands in spite of the presence of respiratory substrates. Since MIA alone does not influence the persistence of oscillations [see note b] irreversible cessation of oscillations shown in Fig.2 points to a lowering of the energetic efficiency of oxidative phosphorylation to a level below that where not only normal contractile behaviour can be maintained but the sample It occurs in spite of the preintegrity as well [notes c and f]. sence of respiration substrates which might be utilized via cytochrome as alternative pathway of electron transport [cp. note d]. Since butyric acid alone does not stop oscillatinos [see Fig.11 the inhibition of mitochondrial functions is not connected with complete inhibition of respiration. Total inhibition of respiration in plasmodia is manifested by reversible cessation of oscillations [cp. note f]. Fig.3 and 4 show that only in case of simultaneous action of butyric acid with SHAM [Fig.31 or KCN !Fig.J] almost complete [Fig.?] or complete [Fig.41 cessation of contraction-relaxation activity is observed. This agrees with the previous conclusion that butyric acid alters mitochondrial functions and on the other hand, also shows that this effect is non-specific in respect of KCN-resistant or KCN-sensitive respiration. It also helps to explain the lack of the stimulation of oscillation with the aid of ketoglutarate or malate with pyruvate [see Fig.21. The return to a normal contractile pattern after washing out butyric acid together with KCN or SHAM [see Fig.3 and 41 shows clearly that in presence of this weak acid glycolysis, in contrast to oxidative
Fig. 3 Reversible salicylhydroxamic
cessation of oscillatins in the presence acid [but]. acid [SHAM] and 10 mM butyric
of 7 mM
Cell Biology
International
Reports,
Vol. 11, No. 4, April
1987
305
Fig. 4 Reversible cessation of the contraction-relaxation cycle in response to 5 mM KCN and 10 mM butyric acid
1
phosphorylation,
? TIME is able
3h
to maintain
the integrity
of the samples.
It is hard to judge whether the inhibition of mitochondrial functions described here is the primary or secondary effect of acidification In any case the influence of butyric acid on of the cytoplasm. mitochondrial activity must be taken into account in the interpretation of the responses of the intact cell to this weak acid. References Baranowski S [1985a]. Alternative pathway of respiration in Physarum polycephalum plasmodia. Cell Biology International Reports 9, 85-90. Baranowski z [1985b]. Consequences of impeding in mitochondrial functions in Physarum polycephalum. III. Reversible cessation of the contraction-relaxation cycle and the temperature sensitivity of the alternate respiratory pathway. European Journal of Cell Biology, 39, 283-289. Cieslawska M, Hrebenda B, and Baranowski Z [1986]. Influence of changes in internal pH on contractile activity in Physarum polycephalum plasmodia. In: Abstracts of 7th European Physarum Meeting University of Kent, 19. Gerson C F, Burton A C [1977]. The relation of cycling of intraCELLULAR PH to mitosis in the acellular slime mould Physarum polycephalum. J Cell Physiol., 91, 297-304. Hirose T, Ueda T and Kobatake Y. [1982]. Changes in intracellular pH accompanying chemoreception in the plasmodia of Physarum polycephalum. J.Gen.Microbiol.128, 2647-2651. Korohoda W, Shraideh Z, Baranowski Z and Wohlfarth-Bottermann K E Energy metabolic regulation of oscillatory contract[1983]. ion activity in Physarum polycephalum. Cell Tiss.Res., 231, 675-691.
Matveeva N B, Beylina S I, Teplov V A and Layrand D B [1978]. Chemotactic and proton responses of the slime mould Physarum polycephalum to non-metabolizable glucose analoques. Acta
Cell Biology
306
International
Reports,
Vol. 11, No. 4, April
1987
Protozool, 18, 173-176. Matveeva N B, Beylina S I, Teplov V A and Layrand D B [1981]. Effect of dicyclohexylcarbodimide on active proton transport and motile behaviour of Physarum polycephalum. In: L Rakoczy ed, Biology of Physarum Proc. Vth European Physarum Meeting, 235-245.
R A [1982]. Changes in intracellular Morisawa M, and Steinhardt pH of Physarum plasmodium during the cell cycle and in response to starvation. Exp. Cell Res., 140, 341-351. Nakamura S, and Kamiya N [1985]. Oscillation of cytoplasmic pH of Physarum plasmodium in relation to motility. Cell Structure and Function, 10, '133-141. Sanders D. and Slayman C L. [1982]. Control of intracellular pII. Predominant role of oxidative metabolism not proton transin the eukaryotic microorganism Neurospora. J.Gen.Phyport, 80, 377-402. siol., Tensiometric demonstration of Wohlfarth-Bottermann K E. [1975]. oscillating contractions in plasmodia of Physarum endogenous, Zeitschrift fur Pflanzenphysiologie, 76, 14polycephalum. 27.
Received
10.11.86
Accepted
3.3.87
International
Reports,
Vol. 11, No. 4, April
301
1987
BUTYRIC ACID AS A SUPRESSOROF MITOCHONDRIAL FUNCTIONS IN PHYSARUM POLYCEPHALUMPLASMODIA M Cieslawska, Nencki
Institute
B Hrebenda
of Experimental Biology, Warsaw, Poland
and Z Baranowski 3 Pasteur
Str.,
02-093
Abstract By means of tensionmetric technique, in the presence of butyric and acid, a comparison is made between the action of respiratory glycolytic inhibitors on plasmodial strands of Physarum polycephalum. It is shown that, in presence of this weak acid, glycolysis but not exidative phosphorylation can preserve the contractile ability of the samples. Inhibitory analysis has revealed that the inhibition of mitochondrial functions by butyric acid is non-specific in the sense that the drug influences both KCN-resistant In all probability the observed and KCN-sensitive respiration. responses of plasmodial strands to butyric acid are related to acidification of the cytoplasm. Introduction It is well established that intracellular pH of a variety of cell the steady-state of proton types is held near pH 7 and represents concentration. In Physarum polycephalum plasmodia internal pH [pHi] undergoes short term oscillations with average value of 6.6 + 0.5 and an amplitude of about 0.1 pH unit [Nakamura and The period of these oscillations coincides with a Kamiya, 19851. minute rhythm of the contraction-relaxation cycle of plasmodial actomyosin. In spite of long term changes of pHi related to mitotic cycle are observed [Gerson and Burton, 1977; Morisawa and Stainhard, 19821. In short term oscillations the phase of acidification coincides with the contraction phase of the contractionrelaxation cycle [Nakamura and Kamiya, 19851. However, the relation between the average value of pHi and contractility or chemotactic behaviour of plasmodia is not yet fully elucidated [Hirose et al., 1982; Matveeva et al., 1979, IgSl]. In the previous report [Cieslawska et al., 19861 we have observed an increase in the frequency of the tensiometrically measured contractile activity of plasmodial strands in the presence of a weak base [procaine] and a decrease in the frequency in response to externally applied weak acid [butyric acid]. These drugs penetrate into the cell in undissociated form and either raise [procaine] or lower [butyric acid] manner [Sanders and Slayman, 1982]. PHi in a concentration-dependent Recent findings concerning the regulation of the contractionrelaxation cycle of plasmodial contractile system [Korohoda et al., 1983; Baranowski, 1985b] allow one to carry out an analysis of the inhibitory influence of any drug on energy-yielding metabolic processes. The results presented in this paper show that the 0309-1651/871040301-06/$03.00/O
0 1987 Academic
Press Inc. (London)
Ltd.
302
Cell Biology
International
Reports,
Vol. 11, No. 4, April
1987
contractile response of plasmodial strand to butyric acid laefLects not cnly the generally accepted effect of decreaeing pH. but also c.auses the inhibition of mitochondrial functions in plaimodia. Material
and Methods
Plasmodial strands isolated from surface cultures of Physarum polycephalum were used for this study. The contractile activity of the strandstis been studied by means of a tension transducer under isometric conditions of measurements [Wohlfarth-Bottermann, 19753. The samples were submerged in salt solution containing: 2mM NaCl, 1 mM KCl, 7 mM MgC12, 1 mM CaC12; pH 5.0-5.2, at a constant temperature of 20°C2 maintained by the use of a Peltier element. The following concentrations of chemicals dissolved in the salt solution were used: 5 mM KCN, ImM iodoacetate [MIA] 10 mM ketoglutaric acid [KG], 10 mM pyruvic acid [pyr], 10 mM L-malic acid [mall, 7 mM salicylhydroxamic acid [SHAM], 10 mM butyric acid [but]. The chemicals were applied externally by replacing solutions surrounding the samples in the perfusion chamber. Results
and Discussion
As was mentioned in the Introduction, butyric acid applied externally lowers the frequency of force oscillations generated by plasmodial strands. To observe this effect [see Fig.11 two preconditions are indispensable: i - the period of oscillations before treatment must be relatively high [2-T min.] and ii - the pH of a solution has to be adjusted near to the value of the pK of butyric This induces a reduction in the internal pH [pHi] of acid [4.8]. The first precondition is the samples [Sanders and Slayman, 19821. The period of oscillthe same as in the case of KCN application. ations in the presence of KCN or butyric acid is about 4 min. inIt suggests that the dependent of its value before treatment. action of butyric acid is not restricted to decreasing pH. but also has an influence on energy metabolism of the plasmodial skrands. The aim of the experiments described in this paper was to check this hvDothesis.
Fig.
decrease of 1. Reversible frequency of force oscillations in response to 10 mM butyric acid [but].
Cell Biology
International
Reports,
Vol. 11, No. 4, April
1987
303
The lack of a reasonable hypothesis concerning processes responsible for oscillatory contraction activity of plasmodial actomyosin does not allow one to interpret unequivically the changes in the period However, the persistence of oscior amplitude of the oscillations. llations or the reversibility of their cessation in response to experimental procedure gives some information about disturbances in This kind of inhibitory analysis is energy-yielding processes. based on following observations previously published [Korohoda et al., 1983; Baranowski, 1985 a,b] [notes a - f]: a
plasmodial mitochondria exhibit pathway, which is KCN-resistant
b
inhibitors of glycolysis [eg. MIA], cytochrome oxidase [KCN], or alternative respiration [eg. SHAM] applied separately, over a time span of a few hours do not show any influence on the persistence of oscillations;
C
an alternative respiratory but SHAM-sensitive;
simultaneous inhibition of glycolysis oxidase [KCN] leads to an irreversible oscillations;
[MIA] and cytochrome cessation of the
d
alternative respiration is able to maintain the oscillations in the presence of KCN with MIA provided that it is supported by externally applied ketoglutarate [KG] or pyruvate with malate [PY~ mall;
e
the oscillations solely maintained by ketoglutarate or pyruvate with malate [ie. in the presence of KCN with MIA] irreversibly disappear after inhibition of the alternative respiratory pathway [SHAM];
f
total inhibition of respiration but not glycolysis [KCN with SHAM treatment] stops the oscillations in a reversible manner although the contractile ability is preserved. Fig. 2 Irreversible cessation of oscillations in response to butyric acid in the presence of iodoacetate [MIA]. FOG concentrations of chemicals and their abbreviations see Material and Methods.
304
Cell Biology
International
Reports,
Vol. 17, No. 4, April
7987
The tensiogram in Fig.2 shows that MIA [an inhibitor of glyceraldehyde-phosphate dehydrogenase] together with butyric acid causes the death of the plasmodial strands in spite of the presence of respiratory substrates. Since MIA alone does not influence the persistence of oscillations [see note b] irreversible cessation of oscillations shown in Fig.2 points to a lowering of the energetic efficiency of oxidative phosphorylation to a level below that where not only normal contractile behaviour can be maintained but the sample It occurs in spite of the preintegrity as well [notes c and f]. sence of respiration substrates which might be utilized via cytochrome as alternative pathway of electron transport [cp. note d]. Since butyric acid alone does not stop oscillatinos [see Fig.11 the inhibition of mitochondrial functions is not connected with complete inhibition of respiration. Total inhibition of respiration in plasmodia is manifested by reversible cessation of oscillations [cp. note f]. Fig.3 and 4 show that only in case of simultaneous action of butyric acid with SHAM [Fig.31 or KCN !Fig.J] almost complete [Fig.?] or complete [Fig.41 cessation of contraction-relaxation activity is observed. This agrees with the previous conclusion that butyric acid alters mitochondrial functions and on the other hand, also shows that this effect is non-specific in respect of KCN-resistant or KCN-sensitive respiration. It also helps to explain the lack of the stimulation of oscillation with the aid of ketoglutarate or malate with pyruvate [see Fig.21. The return to a normal contractile pattern after washing out butyric acid together with KCN or SHAM [see Fig.3 and 41 shows clearly that in presence of this weak acid glycolysis, in contrast to oxidative
Fig. 3 Reversible salicylhydroxamic
cessation of oscillatins in the presence acid [but]. acid [SHAM] and 10 mM butyric
of 7 mM
Cell Biology
International
Reports,
Vol. 11, No. 4, April
1987
305
Fig. 4 Reversible cessation of the contraction-relaxation cycle in response to 5 mM KCN and 10 mM butyric acid
1
phosphorylation,
? TIME is able
3h
to maintain
the integrity
of the samples.
It is hard to judge whether the inhibition of mitochondrial functions described here is the primary or secondary effect of acidification In any case the influence of butyric acid on of the cytoplasm. mitochondrial activity must be taken into account in the interpretation of the responses of the intact cell to this weak acid. References Baranowski S [1985a]. Alternative pathway of respiration in Physarum polycephalum plasmodia. Cell Biology International Reports 9, 85-90. Baranowski z [1985b]. Consequences of impeding in mitochondrial functions in Physarum polycephalum. III. Reversible cessation of the contraction-relaxation cycle and the temperature sensitivity of the alternate respiratory pathway. European Journal of Cell Biology, 39, 283-289. Cieslawska M, Hrebenda B, and Baranowski Z [1986]. Influence of changes in internal pH on contractile activity in Physarum polycephalum plasmodia. In: Abstracts of 7th European Physarum Meeting University of Kent, 19. Gerson C F, Burton A C [1977]. The relation of cycling of intraCELLULAR PH to mitosis in the acellular slime mould Physarum polycephalum. J Cell Physiol., 91, 297-304. Hirose T, Ueda T and Kobatake Y. [1982]. Changes in intracellular pH accompanying chemoreception in the plasmodia of Physarum polycephalum. J.Gen.Microbiol.128, 2647-2651. Korohoda W, Shraideh Z, Baranowski Z and Wohlfarth-Bottermann K E Energy metabolic regulation of oscillatory contract[1983]. ion activity in Physarum polycephalum. Cell Tiss.Res., 231, 675-691.
Matveeva N B, Beylina S I, Teplov V A and Layrand D B [1978]. Chemotactic and proton responses of the slime mould Physarum polycephalum to non-metabolizable glucose analoques. Acta
Cell Biology
306
International
Reports,
Vol. 11, No. 4, April
1987
Protozool, 18, 173-176. Matveeva N B, Beylina S I, Teplov V A and Layrand D B [1981]. Effect of dicyclohexylcarbodimide on active proton transport and motile behaviour of Physarum polycephalum. In: L Rakoczy ed, Biology of Physarum Proc. Vth European Physarum Meeting, 235-245.
R A [1982]. Changes in intracellular Morisawa M, and Steinhardt pH of Physarum plasmodium during the cell cycle and in response to starvation. Exp. Cell Res., 140, 341-351. Nakamura S, and Kamiya N [1985]. Oscillation of cytoplasmic pH of Physarum plasmodium in relation to motility. Cell Structure and Function, 10, '133-141. Sanders D. and Slayman C L. [1982]. Control of intracellular pII. Predominant role of oxidative metabolism not proton transin the eukaryotic microorganism Neurospora. J.Gen.Phyport, 80, 377-402. siol., Tensiometric demonstration of Wohlfarth-Bottermann K E. [1975]. oscillating contractions in plasmodia of Physarum endogenous, Zeitschrift fur Pflanzenphysiologie, 76, 14polycephalum. 27.
Received
10.11.86
Accepted
3.3.87