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Depolarization of the mitochondrial membrane potential increases free cytosolic calcium in synaptosomes
Neuroscience Letters, 49 (1984) 33-37
33
Elsevier Scientific Publishers Ireland Ltd. NSL 02838
DEPOLARIZATION OF THE MITOCHONDRIAL MEMBRANE POTENTIAL INCREASES FREE CYTOSOLIC CALCIUM IN SYNAPTOSOMES
ERKKI HEINONEN l'*, KARL E.O. ,AKERMAN2 and KAI KAILA 3
Department of Physiology, University of Helsinki; 2 fiibo A kademi, Department of Biochemistry and Pharmacy," and 3 Department of Zoology, Division of Physiology, University of Helsinki, Helsinki (Finland) (Received April 12th, 1984; Accepted May 14th, 1984)
Key words: synaptosomes - mitochondria - cytosolic calcium - arsenazo Ill - carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP)
Intracellular calcium transients in synaptosomes, isolated from the guinea pig brain, were measured using entrapped metallochromic indicator arsenazo III. Addition of 1 #M carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) increased rapidly the absorbance of the entrapped arsenazo III, indicating an increase in the cytosolic free calcium. The FCCP-induced increase in cytoplasmic free Ca 2+ was not blocked by 200/tM verapamil, while the increment in calcium caused by 40/zM veratridine was verapamil-sensitive. The absorbance changes induced by FCCP were not significantly increased when the extracellular potassium concentration was elevated from 5.4 to 50 mM. These data indicate that in nerve endings of mammalian brain, cytoplasmic free calcium, which is essential for the release of transmitter, is increased on depolarization of major intracellular calcium buffers, mitochondria.
The role of an increase in cytosolic Ca 2 + in transmitter liberation from nerve terminals is well known [6]. Far less is known about mechanisms of fine control of intraterminal Ca 2 + in events such as facilitation or termination of transmission. Alnaes and R a h a m i m o f f [8] have demonstrated a facilitation of neuromuscular transmission by mitochondrial uncoupling agents and they suggested a role of internal mitochondria in regulating cytosolic Ca 2+ . Studies employing 45Ca2 + have demonstrated that a main fraction of the calcium taken up by isolated nerve endings upon depolarization resides in intrasynaptosomal mitochondria (for reviews see refs. 7 and 11). Uncouplers of oxidative phosphorylation depolarize the mitochondrial m e m b r a n e [14] and release Ca 2 ÷ from mitochondria of nerve endings in situ [2,15]. Studies employing 45Ca 2 ÷ do not, however, give information about the actual change in free Ca 2 ÷ within nerve endings, since other Ca 2 ÷ buffering systems * Author for correspondence at: Department of Physiology, University of Helsinki, Siltavuorenpenger 20 J, SF-00170 Helsinki 17, Finland.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
34
m a y compensate for a change in cytosolic Ca 2 ÷ upon release from mitochondria. We have recently described a method for entrapping a Ca 2 ÷ indicator, arsenazo III, within isolated nerve endings [1]. The aim of the present study was to investigate the role of mitochondria in regulating the cytosolic Ca 2 ÷ of isolated nerve endings by employing the above-mentioned method. The procedure for isolation of the guinea pig cerebral cortical synaptosomes containing entrapped arsenazo III and the materials and methods used were as described previously [1]. After isolation the synaptosomes were resuspended in fresh standard N a ÷ - b a s e d medium (137 m M NaC1-5.4 m M K C I - I . 2 m M MGC12-0.44 mM KHzPO4-4.2 mM NaHCO3-20 mM Tes, pH 7.4) to a protein concentration of 1.5-3 m g / l for spectrophotometry. As demonstrated previously [1], the addition of veratridine, which opens Na ÷ channels in the plasma membrane and thus depolarizes synaptosomes, induced an increase in the absorbance of entrapped arsenazo III indicating a rise in cytosolic Ca 2 + (Fig. 1). A further increment in absorbance occurred upon addition of FCCP, which depolarizes mitochondria. Oligomycin was added simultaneously in order to prevent A T P depletion due to activation of the mitochondrial ATPase by the uncoupler [3]. The saturation of the response with ionophore A23187, a 2H ÷ / C a 2 ÷ exchanger, indicates the saturation of the arsenazo III to Ca 2 ~ , which occurs at a Ca 2÷ around 100 #M [1,13]. When F C C P was added prior to veratridine a
0,002
2:3
CP
lOOs '
r
ver
ver
FCCP
Fig. 1. Effects of F C C P , veratridine and A23187 on the absorbance of entrapped arsenazo 1II. Synaptosomal protein was 3 m g / m l and extracellular CaCI2 was 1 raM. The changes in absorbance at the wavelength pair of 600/555 n m were monitored. 1/~M F C C P together with 4/~g/ml oligomycin (FCCP), 40 #M veratridine (ver) and 4/~M A23187 (A23) were added as indicated.
35
somewhat smaller response to this agent was seen. Note that the total absorbance change was of similar magnitude in every recording, suggesting that the absorbance responses to the uncoupler indeed represent a change in cytosolic Ca 2 +. The effect of FCCP was unaffected by verapamil, while the response to a subsequent addition of veratridine was inhibited (Fig. 2). Since verapamil blocks Ca 2 ÷ channels of nerve endings [1,5], this finding excludes a possible activation of Ca 2 ÷ channels of the plasma membrane by the uncoupler. When the extracellular potassium concentration was elevated from 5.4 to 50 mM the corresponding saturable absorbance response obtained with A23187 decreased 24°70 (results from five experiments) due to the additional Ca 2 ÷ influx induced by high-K + depolarization. During high-K ÷ depolarization the mean increase in absorbance changes induced by 1 #M FCCP was 5070 higher when compared to the corresponding changes during control conditions. It is known that high-K ÷ depolarization considerably increases both synaptosomal calcium content and mitochondrial Ca 2 ÷ accumulation in situ [2]. Thus the apparently small increase in cytosolic Ca 2 + induced by the uncoupler in K +-depolarized synaptosomes indicates that the excess of Ca 2 + released
f A23 ver FCCP
ver
A23
o.o I
1! min ! Fig. 2. Effect of verapamil on FCCP- and veratridine-induced increase in cytosolic Ca 2 +. Conditions as in Fig. 1. The synaptosomal protein was 1.5 mg/ml. Additions were made of 1 /zM FCCP, 40 #M veratridine (ver) and 4 ttM A23187 (A23) as indicated. In the lower trace 200/~M verapamil were added just before the recording.
36 by F C C P from mitochondria is rapidly pumped out across the plasma membrane. This is in accordance with the results obtained by Akerman and Nicholls [2]. The results of the present study show qualitatively that cytosolic free Ca 2 + in synaptosomes is increased in response to the uncoupler FCCP. The increase in Ca 2 ~ is not due to an interference with A T P production since glycolysis is able to maintain high A T P levels in similar conditions for at least 10 min [3]. It has been demonstrated [9] that in squid giant axons the increase in cytosolic free Ca 2 ÷ induced by uncouplers is much larger in stimulated, i.e. Ca 2 ÷ loaded, than in resting axons. This appears not to be the case with synaptosomes. The difference might be due to the higher ratio of mitochondrial versus cytoplasmic volume in nerve endings. There is evidence that increased levels of cytosolic Ca 2 + facilitate the release o f transmitter [10] and that uncouplers increase transmitter liberation at the neuromuscular junction [8]. In the synaptosomal preparation the Ca 2 + dependency of transmitter release closely parallels the activity of the mitochondrial Ca 2 ÷ uptake pathway [4], and the present experiments show that ionized free Ca 2 ÷ can be increased by release from the mitochondrial pool. Mitochondria have a rapid electrophoretic uptake mechanism for Ca 2 + and a comparably slower electroneutral Ca 2 + release mechanism (for review see ref. 7). However, there is no clearcut evidence for physiological mechanisms that release Ca 2 + from mitochondria during the electrical activity of nerve terminals, for discussion see refs. 5 and 12. It does not, however, seem unreasonable to suggest that mitochondria play a role in transmission by limiting the rise in cytosolic Ca 2 + . On the other hand, a slow release of Ca 2 + from the mitochondrial pool might represent a part of residual Ca 2 ÷ inducing potentiation of the release of transmitter. This study was aided by grants from the Academy of Finland and the Sigrid Jus61ius Foundation. 1 ,~kerman, K.E.O. and Heinonen, E., Qualitative measurements of cytosolic calcium ion concentration within isolated guinea pig nerve endings using entrapped arsenazo IIl, Biochim. Biophys. Acta, 732 (1983) 117-121. 2 Akerman, K.E.O. and Nicholls, D.G.,, lntrasynaptosomal compartmentation of calcium during depolarization-induced calcium uptake across the plasma membrane, Biochim. Biophys. Acta, 645 (1981) 41-48. 3 Akerman, K.E.O. and Nicholls, D.G., ATP depletion increases Ca 2 + uptake by synaptosomes, FEBS Lett., 135 (1981) 212-214. 4 A.kerman, K.E.O. and Nicholls, D.G., Calcium transport by intact synaptosomes: influence of ionophore A23187 on plasma-membrane potential, plasma-membrane calcium transport, mitochondrial membrane potential, respiration, cytosolic free-calcium concentration and noradrenaline release, Europ. J. Biochem., 115 (1981)67-73. 5 Akerman, K.E.O. and Nicholls, D.G., Ca 2+ transport by intact synaptosomes: the voltagedependent Ca 2 ÷ -channel and a re-evaluation of the role of sodium/calcium exchange, Europ. J. Biochem., 117 (1981) 491-497. 6 /kkerman, K.E.O. and Nicholls, D.G., Ca 2 + transport and the regulation of transmitter release in isolated nerve endings, TIBS, 8 (1983) 63-64.
37 7 /kkerman, K.E.O and Nicholls, D.G., Physiological and bioenergetic aspects of mitochondrial calcium transport, Rev. Physiol. Biochem. Pharmacol., 95 (1983) 149-201. 8 Alneas, E. and Rahamimoff, R., On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol. (Lond.), 248 (1975) 285-306. 9 Brinley, F.J., Tiffert, T., Scarpa, A. and Mullins, L.J., Intracellular buffering capacity in isolated squid axons, J. gen. Physiol., 70 (1977) 355-384. 10 Charlton, M.P., Smith, S.J. and Zucker, R.S., Role of presynaptic calcium ions and channels in synaptic facilitation and depression at the giant squid synapse, J. Physiol. (Lond.), 323 (1982) 173-193. 11 Nicholls, D.G. and Akerman, K.E.O., Biochemical approaches to the study of cytosolic calcium regulation in nerve endings, Phil. Trans. Soc. Lond. B, 296 (1981) 115-122. 12 Rahamimoff, R., Lev-Tov, A. and Meiri, H., Primary and secondary regulation of quantal transmitter release: calcium and sodium, J. exp. Biol., 89 (1980) 5-18. 13 Scarpa, A., Measurements of cation transport with metallochromic indicators, Meth. Enzymol., 56 (1979) 301-339. 14 Scott, I.D. and Nicholls, D.G., Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ, Biochem. J., 186 (1980) 21-33. 15 Scott, I.D., .~kerman, K.E.O. and Nicholls, D.G., Calcium-ion transport by intact synaptosomes: intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential, Biochem. J., 192 (1980) 873-880.
33
Elsevier Scientific Publishers Ireland Ltd. NSL 02838
DEPOLARIZATION OF THE MITOCHONDRIAL MEMBRANE POTENTIAL INCREASES FREE CYTOSOLIC CALCIUM IN SYNAPTOSOMES
ERKKI HEINONEN l'*, KARL E.O. ,AKERMAN2 and KAI KAILA 3
Department of Physiology, University of Helsinki; 2 fiibo A kademi, Department of Biochemistry and Pharmacy," and 3 Department of Zoology, Division of Physiology, University of Helsinki, Helsinki (Finland) (Received April 12th, 1984; Accepted May 14th, 1984)
Key words: synaptosomes - mitochondria - cytosolic calcium - arsenazo Ill - carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP)
Intracellular calcium transients in synaptosomes, isolated from the guinea pig brain, were measured using entrapped metallochromic indicator arsenazo III. Addition of 1 #M carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) increased rapidly the absorbance of the entrapped arsenazo III, indicating an increase in the cytosolic free calcium. The FCCP-induced increase in cytoplasmic free Ca 2+ was not blocked by 200/tM verapamil, while the increment in calcium caused by 40/zM veratridine was verapamil-sensitive. The absorbance changes induced by FCCP were not significantly increased when the extracellular potassium concentration was elevated from 5.4 to 50 mM. These data indicate that in nerve endings of mammalian brain, cytoplasmic free calcium, which is essential for the release of transmitter, is increased on depolarization of major intracellular calcium buffers, mitochondria.
The role of an increase in cytosolic Ca 2 + in transmitter liberation from nerve terminals is well known [6]. Far less is known about mechanisms of fine control of intraterminal Ca 2 + in events such as facilitation or termination of transmission. Alnaes and R a h a m i m o f f [8] have demonstrated a facilitation of neuromuscular transmission by mitochondrial uncoupling agents and they suggested a role of internal mitochondria in regulating cytosolic Ca 2+ . Studies employing 45Ca2 + have demonstrated that a main fraction of the calcium taken up by isolated nerve endings upon depolarization resides in intrasynaptosomal mitochondria (for reviews see refs. 7 and 11). Uncouplers of oxidative phosphorylation depolarize the mitochondrial m e m b r a n e [14] and release Ca 2 ÷ from mitochondria of nerve endings in situ [2,15]. Studies employing 45Ca 2 ÷ do not, however, give information about the actual change in free Ca 2 ÷ within nerve endings, since other Ca 2 ÷ buffering systems * Author for correspondence at: Department of Physiology, University of Helsinki, Siltavuorenpenger 20 J, SF-00170 Helsinki 17, Finland.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
34
m a y compensate for a change in cytosolic Ca 2 ÷ upon release from mitochondria. We have recently described a method for entrapping a Ca 2 ÷ indicator, arsenazo III, within isolated nerve endings [1]. The aim of the present study was to investigate the role of mitochondria in regulating the cytosolic Ca 2 ÷ of isolated nerve endings by employing the above-mentioned method. The procedure for isolation of the guinea pig cerebral cortical synaptosomes containing entrapped arsenazo III and the materials and methods used were as described previously [1]. After isolation the synaptosomes were resuspended in fresh standard N a ÷ - b a s e d medium (137 m M NaC1-5.4 m M K C I - I . 2 m M MGC12-0.44 mM KHzPO4-4.2 mM NaHCO3-20 mM Tes, pH 7.4) to a protein concentration of 1.5-3 m g / l for spectrophotometry. As demonstrated previously [1], the addition of veratridine, which opens Na ÷ channels in the plasma membrane and thus depolarizes synaptosomes, induced an increase in the absorbance of entrapped arsenazo III indicating a rise in cytosolic Ca 2 + (Fig. 1). A further increment in absorbance occurred upon addition of FCCP, which depolarizes mitochondria. Oligomycin was added simultaneously in order to prevent A T P depletion due to activation of the mitochondrial ATPase by the uncoupler [3]. The saturation of the response with ionophore A23187, a 2H ÷ / C a 2 ÷ exchanger, indicates the saturation of the arsenazo III to Ca 2 ~ , which occurs at a Ca 2÷ around 100 #M [1,13]. When F C C P was added prior to veratridine a
0,002
2:3
CP
lOOs '
r
ver
ver
FCCP
Fig. 1. Effects of F C C P , veratridine and A23187 on the absorbance of entrapped arsenazo 1II. Synaptosomal protein was 3 m g / m l and extracellular CaCI2 was 1 raM. The changes in absorbance at the wavelength pair of 600/555 n m were monitored. 1/~M F C C P together with 4/~g/ml oligomycin (FCCP), 40 #M veratridine (ver) and 4/~M A23187 (A23) were added as indicated.
35
somewhat smaller response to this agent was seen. Note that the total absorbance change was of similar magnitude in every recording, suggesting that the absorbance responses to the uncoupler indeed represent a change in cytosolic Ca 2 +. The effect of FCCP was unaffected by verapamil, while the response to a subsequent addition of veratridine was inhibited (Fig. 2). Since verapamil blocks Ca 2 ÷ channels of nerve endings [1,5], this finding excludes a possible activation of Ca 2 ÷ channels of the plasma membrane by the uncoupler. When the extracellular potassium concentration was elevated from 5.4 to 50 mM the corresponding saturable absorbance response obtained with A23187 decreased 24°70 (results from five experiments) due to the additional Ca 2 ÷ influx induced by high-K + depolarization. During high-K ÷ depolarization the mean increase in absorbance changes induced by 1 #M FCCP was 5070 higher when compared to the corresponding changes during control conditions. It is known that high-K ÷ depolarization considerably increases both synaptosomal calcium content and mitochondrial Ca 2 ÷ accumulation in situ [2]. Thus the apparently small increase in cytosolic Ca 2 + induced by the uncoupler in K +-depolarized synaptosomes indicates that the excess of Ca 2 + released
f A23 ver FCCP
ver
A23
o.o I
1! min ! Fig. 2. Effect of verapamil on FCCP- and veratridine-induced increase in cytosolic Ca 2 +. Conditions as in Fig. 1. The synaptosomal protein was 1.5 mg/ml. Additions were made of 1 /zM FCCP, 40 #M veratridine (ver) and 4 ttM A23187 (A23) as indicated. In the lower trace 200/~M verapamil were added just before the recording.
36 by F C C P from mitochondria is rapidly pumped out across the plasma membrane. This is in accordance with the results obtained by Akerman and Nicholls [2]. The results of the present study show qualitatively that cytosolic free Ca 2 + in synaptosomes is increased in response to the uncoupler FCCP. The increase in Ca 2 ~ is not due to an interference with A T P production since glycolysis is able to maintain high A T P levels in similar conditions for at least 10 min [3]. It has been demonstrated [9] that in squid giant axons the increase in cytosolic free Ca 2 ÷ induced by uncouplers is much larger in stimulated, i.e. Ca 2 ÷ loaded, than in resting axons. This appears not to be the case with synaptosomes. The difference might be due to the higher ratio of mitochondrial versus cytoplasmic volume in nerve endings. There is evidence that increased levels of cytosolic Ca 2 + facilitate the release o f transmitter [10] and that uncouplers increase transmitter liberation at the neuromuscular junction [8]. In the synaptosomal preparation the Ca 2 + dependency of transmitter release closely parallels the activity of the mitochondrial Ca 2 ÷ uptake pathway [4], and the present experiments show that ionized free Ca 2 ÷ can be increased by release from the mitochondrial pool. Mitochondria have a rapid electrophoretic uptake mechanism for Ca 2 + and a comparably slower electroneutral Ca 2 + release mechanism (for review see ref. 7). However, there is no clearcut evidence for physiological mechanisms that release Ca 2 + from mitochondria during the electrical activity of nerve terminals, for discussion see refs. 5 and 12. It does not, however, seem unreasonable to suggest that mitochondria play a role in transmission by limiting the rise in cytosolic Ca 2 + . On the other hand, a slow release of Ca 2 + from the mitochondrial pool might represent a part of residual Ca 2 ÷ inducing potentiation of the release of transmitter. This study was aided by grants from the Academy of Finland and the Sigrid Jus61ius Foundation. 1 ,~kerman, K.E.O. and Heinonen, E., Qualitative measurements of cytosolic calcium ion concentration within isolated guinea pig nerve endings using entrapped arsenazo IIl, Biochim. Biophys. Acta, 732 (1983) 117-121. 2 Akerman, K.E.O. and Nicholls, D.G.,, lntrasynaptosomal compartmentation of calcium during depolarization-induced calcium uptake across the plasma membrane, Biochim. Biophys. Acta, 645 (1981) 41-48. 3 Akerman, K.E.O. and Nicholls, D.G., ATP depletion increases Ca 2 + uptake by synaptosomes, FEBS Lett., 135 (1981) 212-214. 4 A.kerman, K.E.O. and Nicholls, D.G., Calcium transport by intact synaptosomes: influence of ionophore A23187 on plasma-membrane potential, plasma-membrane calcium transport, mitochondrial membrane potential, respiration, cytosolic free-calcium concentration and noradrenaline release, Europ. J. Biochem., 115 (1981)67-73. 5 Akerman, K.E.O. and Nicholls, D.G., Ca 2+ transport by intact synaptosomes: the voltagedependent Ca 2 ÷ -channel and a re-evaluation of the role of sodium/calcium exchange, Europ. J. Biochem., 117 (1981) 491-497. 6 /kkerman, K.E.O. and Nicholls, D.G., Ca 2 + transport and the regulation of transmitter release in isolated nerve endings, TIBS, 8 (1983) 63-64.
37 7 /kkerman, K.E.O and Nicholls, D.G., Physiological and bioenergetic aspects of mitochondrial calcium transport, Rev. Physiol. Biochem. Pharmacol., 95 (1983) 149-201. 8 Alneas, E. and Rahamimoff, R., On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol. (Lond.), 248 (1975) 285-306. 9 Brinley, F.J., Tiffert, T., Scarpa, A. and Mullins, L.J., Intracellular buffering capacity in isolated squid axons, J. gen. Physiol., 70 (1977) 355-384. 10 Charlton, M.P., Smith, S.J. and Zucker, R.S., Role of presynaptic calcium ions and channels in synaptic facilitation and depression at the giant squid synapse, J. Physiol. (Lond.), 323 (1982) 173-193. 11 Nicholls, D.G. and Akerman, K.E.O., Biochemical approaches to the study of cytosolic calcium regulation in nerve endings, Phil. Trans. Soc. Lond. B, 296 (1981) 115-122. 12 Rahamimoff, R., Lev-Tov, A. and Meiri, H., Primary and secondary regulation of quantal transmitter release: calcium and sodium, J. exp. Biol., 89 (1980) 5-18. 13 Scarpa, A., Measurements of cation transport with metallochromic indicators, Meth. Enzymol., 56 (1979) 301-339. 14 Scott, I.D. and Nicholls, D.G., Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ, Biochem. J., 186 (1980) 21-33. 15 Scott, I.D., .~kerman, K.E.O. and Nicholls, D.G., Calcium-ion transport by intact synaptosomes: intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential, Biochem. J., 192 (1980) 873-880.