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ATP-stimulated glutamate-dependent calcium uptake by rat synaptosomes
Clinica Chimiea Acta, 206 (1992) 147-153 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8983/92605.00
147
CCA 05254
ATP-stimulated glutamate-dependent uptake by rat synaptosomes
calcium
L. Galzigna, M. Bianchi, T, Battistin, M. Scarpa and A. Rigo Department of Biological Chemistry, University of Padova. 35121 Padova (Italy)
(Received 28 October 1991; accepted 23 November 1991) Key words: Extracellular ATP: Synaptosomes; Glutamate; Calcium
Smnmary The entry of Ca2+ in rat synaptosomes was followed with a Ca2+-selective electrode. Extracellular ATP is necessary for the entry which is a function of synaptosomal protein, free Ca2+ and glutamate concentrations. Ketamine, glycine and kainate have negligible effect while quisqualate slightly inhibits the uptake of Ca2+ in the presence of glutamate. The added ATP is hydrolyzed by the synaptosomes through an ouabain-insensitive ecto-ATPase affected by the presence of Ca2+, glutamate and, to a slight extent, NMDA.
Introduction Glutamate is the major neuroexcitatory transmitter in the central nervous system and different types of receptors are capable of binding glutamate or its agonists (e.g. kainate, quisqualate, ~-methyl-~-aspa~ate or NMDA) and are modulated by effectors such as glycine, ketamine or magnesium f 11. The binding of glutamate to the postsynaptic membrane induces the opening of calcium channels and the latter event initiates a cascade of second messengers acting as transducers of the excitatory signal. Glutamate has been shown to stimulate the entry of calcium into neural cells through a channel associated with the NMDA receptor and at least another voltageoperated channel opened up by the ~utamate-indu~ de~la~zation of the membrane [ 21. This mechanism is of an outstanding pathophysiological significance and, after having studied its relationship with the process of glutamate uptake [ 3 I, i.e. the main system utilized for the intrasynaptic termination of the action of glutamate, we concentrated on the measurement of calcium entrance into synaptosomes in the presence of extracellular ATP. ATP is known to stimulate the uptake Correspondence fo: L. Galzigna, Ph.D., M.D. Professor of Biochemistry, Department of Biological Chemistry, via Trieste 75, 35121 Padova, Italy.
148
of glutamate in isolated synaptic vesicles [ 41 and a coupling of such an uptake with the hydrolysis of ATP by a proton-pumping ATPase has been demonstrated [ 5 J . Purinergic receptors on the outer face of the cell membrane, on the other hand, seem to be quite diffused [6] and the binding of purine nucleotides is generally related with changes of ionic translocation through the membranes. Hereby we report our results on the uptake of calcium by rat synaptosomes in the presence of glutamate and with the addition of ATP. The entrance of calcium was followed, under different conditions, with an ion-selective electrode, and the hydrolysis of ATP was monitored by a 3’P-NMR technique. Materials and Methods Chemicals and instruments Ketamine (Ketalar) was obtained from Parke-Davis, Milan. All other chemicals were purchased from Sigma Chem. Co. (St. Louis, USA). Ca*+ was detected by a Ca-selective electrode by using a calomel-electrode as a reference (Metrohm AC, Herisau, CH). Synaptosomes Cerebral.synaptosomes were isolated from Wistar male rats (300-350 g body wt.) according to Nicholls [7] by using a discontinuous Ficoll density gradient. Protein was measured by the method of Lowry et al. [ 8 ] with bovine serum albumin as a standard. The synaptosomes, after separation in the gradient by centrifugation at 70,000 x g for 30 min in a Sorvall ultracentrifuge were stored in ice and all experiments were carried out within 2 h from the preparation. Ca2+ uptake assay Synaptosomal protein (0.75 mg) was preincubated with 2 mM ATP for 5 min at 23°C and then resuspended in 3 ml of a medium composed of 5 mM Tris-HCI, 4 mM MgS04, 0,25 M sucrose at a final pH of 7.4, unless otherwise stated. Some experiments were in fact carried out with different times of pre-incubation with ATP or different protein concentration. The amount of free Ca*+ detectable at different times after the addition of the synaptosomes/ATP to the medium was calculated on the basis of calibration curves carried out in the different conditions employed to transform the recorded mV values into Ca2+ concentration. ATP hydrolysis by 3’P-NMR Synaptosomal protein (2 mg) was resuspended in 2.5 ml of a medium composed of 50 mM 2-(2-hydroxy-l,l-bis(hydroxymethyl)-ethyl)-amino-ethanesulphonate, 4 mM MgS04, 0.5 mM ouabain and 0.25 M sucrose at a final pH of 7.4 with 4 mM tetraphenylphosphonium chloride as a reference. After 5 min of preincubation 5 mM ATP sodium salt neutralized with T&base was added. The resolution time, under
149
those conditions, was 1.5 min and the experiments were carried out in 10 mm tubes with a spinning rate of 10 Hz by using automated microprograms. The spectra were recorded on a Bruker MSL 300 NMR spectrometer operating at 121 MHz. Results We first checked the progressive decrease of free Ca’+, i.e. the increase of Ca*+ entry, with increasing concentration of synaptosomal protein (Fig. 1). Note that the experiments were carried out in the presence of 4 mM Mg*+ and since ATP was 2 mM there was an excess of free Mg *+ which is known to block the NMDA channel ill. In the absence of ATP no synaptosomal Ca*+ uptake was observed, while in the presence of ATP the extent of the uptake seems to increase with increasing times of preincubation of the synaptosomes with ATP (Fig. 2). Table I shows the kinetic of Ca*+ uptake expressed as mV values recorded at different times. The uptake of Ca*+ is influenced to a different extent by increasing concentrations of glutamate and NMDA (Table II). Increasing concentrations of glutamate in fact progressively stimulate the Ca2+ entry, while increasing concentrations of NMDA have, all in all, negligible effects. The amount of free Ca*+ seems to be crucial for the Ca*+ uptake
1
2 [PROTEIN] (mg)
3
Fig. 1. Effect of increasing protein concentration on the synaptosomal uptake of Ca2+. Conditions as in Materials and Methods. The preincubation time with ATP was 5 min. CaZ+ was 5 x 10” M. Means f SD. of 4 experiments.
150
\ .-C E \
f4
d E
.6 -
5
10
PREINCUBATION
15 TIME
20 (mid
Fig. 2. Effect of the times of preincubation with ATP on the uptake of Ca*+ by synaptosomes. Conditions as in Materials and Methods. Ca*+ was 5 x 10m6M. Means * SD. of 4 experiments.
process (Fig. 3); the concentration of free Ca2+ is related to the amount of bound Ca2+ by a saturation kinetic and a Scatchard’s type plot of Fig. 3 data (not shown) is linear suggesting the existence of one class of Ca2+ binding sites on the synaptosomal membrane. The action of agonists and effecters is summarized in Table III showing that the Ca2+ uptake is slightly inhibited by quisqualate while the other compounds seem uneffective. This was confirmed with higher concentrations of the effecters (not shown). The kinetics of ATP hydrolysis, followed by the disappearance of the P-ATP
TABLE I Kinetic of Ca*+ entry into synaptosomes. Ca*+ was IO-’ M and the conditions are described in Materials and Methods. Values are mean f SD. of 4 experiments. Addition (times)
mV
Medium Medium Medium Medium Medium
-5.0 -10.3 -15.0 -19.6 -23.9
+ + + + +
Ca2+ Synaptosomes/ATP Synaptosomes/ATP (1 min) Synaptosomes/ATP (2 min) SynaptosomesIATP (3 min)
mV f 0.05 f 0.08 l 0.12 f 0.11 f 0.17
-5.3 -10.0 -14.6 -18.9
TABLE II Effect of glutamate and NMDA on Ca2+ synaptosomal uptake. The values are means + S.D. from 5 experiments. Ca2+ concentration was 5 x lOm6M and a 100% value corresponds to 0.32 i: 0.08 gmol Ca2+/min/mg protein. Addition
Glutamate
NMDA
None 100 10e5 M Glutamate or NMDA 10V4M Glutamate or NMDA 5 x 10m4M Glutamate or NMDA 10V3M Glutamate or NMDA
107 135 214 200
-
-6
f f f f
5 6 8 8
91 II3 101 83
f f f f
3 5 4 5
-3 fmolll)
Fig. 3. Effect of increasing concentrations of free Ca 2c on the synaptosomal uptake of Ca*+. Conditions as in Materials and Methods. Means f SD. of 4 experiments.
152 TABLE III Synaptosomal Ca2+ uptake in the presence of effecters. The values are means f S.D. of 4 experiments; Ca2+ was 5 x low6 M and glutamate 10m4 M. A 100% value corresponds to 0.42 f 0.04 pmol Ca2+/min/mg protein. Addition
Percent
None 10m4Ketamine 10m4Quisqualate 10e4 Kainate 10e4 Glycine
100 98 80 102 93
* ze f f
2 5 6 3
signal with the 3’P-NMR technique, is faster in the presence of glutamate, Cal+ and, to a lower extent, NMDA (Table IV).
The present data are in full agreement with those obtained by using a completely different technique, i.e. the measurement of the residual radioactivity left within the synaptosomes after filtration through Millipore of a synaptosomal suspension incubated with 45Ca [ 31. Glutamate and ATP activate the uptake of Ca*+; ketamine, a dissociative anesthetic used in clinical practice, is bound to the NMDA receptorEa*+ channel and the same is true for glycine, while kainate and quisqualate are bound to other glutamate receptors. The present results suggest that the glutamate-dependent Ca*+ uptake is a rapid process since the instrumental response is immediate and linear up to 3 min from the addition of the synaptosomes. The most likely involved transport system seems to be the voltage-operated channel which is known to respond to the glutamateinduced depolarization [ 21. The usual effecters of the NMDA receptor-linked channel are in fact without any effect on the process. The ouabain-insensitive ATP hydrolysis is stimulated to a different extent by the presence of glutamate, Ca*+ and
TABLE IV ATP hydrolysis followed by the 31P-NMR technique (means f S.D. of 3 experiments) Addition
nmol ATP hydrolyzed/min/mg protein
None +4 mM Ca2+ +4 mM Glutamate +4 mM NMDA
137 287 233 160
ziz 25 zt 71 f 61 f 66
153
NMDA and this agrees with the respective effects of glutamate, ATP and NMDA on Ca2+ entry. Evidently any attempt at suggesting the clinical implications of the present findings is farfetched, although the importance of a control of the neuroexcitato~ transmission is undisputed for both normal and pathological functioning of the central nervous system. It is known that extracellular ATP can influence, at micromolar concentration, processes such as platelet aggregation, vascular tone, cardiac function, muscle contraction and, in addition, both peripheral and central neurotransmission [ 61. Nucleotides can in fact be released from neural cells by exocytosis and are metabolized by ecto-nucleotidases of which the ouabain-insensitive ATPase of this paper may be an example. The concept of ATP as a purinergic transmitter has been put forward, together with that of its neuroeffector and neuromodulator activities [ 61 but more work has to be done before these ideas can find a clinical application.
This research has been carried out with the financial help of the Ministry of Education (grant 06/89) and the Regione Veneto (grant 200/88). References 1 2 3 4 5 6 7 8
Cotman CW, Monaghan DT. Excitatory neurotransmission: NMDA receptors and Hebb-type synaptic plasticity. Ann Rev Neurosci 1988:!I:61 -80. Mayer ML, Miller RJ. Excitatory aminoacid receptors, second messengers and regulation of intracellular Ca*+ in mammalian neurons. Trends Pharmacol Sci 1990;11:254-260. Galzigna L, Bianchi M, Rizzo V. Battistin T, Scarpa M, Rigo A. ATFstimulated uptake of calcium and glutamate in rat synaptosomes. Cell B&hem. & Function 1992; in press. Naito S, Ueda T. Adenosine triphosphate-dependent uptake of glutamate into protein I-associated synaptic vesicles. J Biol Chem 1983;259:696-699. Shioi J, Naito S, Ueda T. Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane. Biochem J 1989;258:499-504. Gordon JL. Extracellular ATP:effects, sources and fate. Biochem J 1986;233:309-319. Nicholls DG. Calcium transport and proton electrochemical potential gradient in mitochondria from guinea pig cerebral cortex and rat heart. Biochem J 1978;170:511-522. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275.
147
CCA 05254
ATP-stimulated glutamate-dependent uptake by rat synaptosomes
calcium
L. Galzigna, M. Bianchi, T, Battistin, M. Scarpa and A. Rigo Department of Biological Chemistry, University of Padova. 35121 Padova (Italy)
(Received 28 October 1991; accepted 23 November 1991) Key words: Extracellular ATP: Synaptosomes; Glutamate; Calcium
Smnmary The entry of Ca2+ in rat synaptosomes was followed with a Ca2+-selective electrode. Extracellular ATP is necessary for the entry which is a function of synaptosomal protein, free Ca2+ and glutamate concentrations. Ketamine, glycine and kainate have negligible effect while quisqualate slightly inhibits the uptake of Ca2+ in the presence of glutamate. The added ATP is hydrolyzed by the synaptosomes through an ouabain-insensitive ecto-ATPase affected by the presence of Ca2+, glutamate and, to a slight extent, NMDA.
Introduction Glutamate is the major neuroexcitatory transmitter in the central nervous system and different types of receptors are capable of binding glutamate or its agonists (e.g. kainate, quisqualate, ~-methyl-~-aspa~ate or NMDA) and are modulated by effectors such as glycine, ketamine or magnesium f 11. The binding of glutamate to the postsynaptic membrane induces the opening of calcium channels and the latter event initiates a cascade of second messengers acting as transducers of the excitatory signal. Glutamate has been shown to stimulate the entry of calcium into neural cells through a channel associated with the NMDA receptor and at least another voltageoperated channel opened up by the ~utamate-indu~ de~la~zation of the membrane [ 21. This mechanism is of an outstanding pathophysiological significance and, after having studied its relationship with the process of glutamate uptake [ 3 I, i.e. the main system utilized for the intrasynaptic termination of the action of glutamate, we concentrated on the measurement of calcium entrance into synaptosomes in the presence of extracellular ATP. ATP is known to stimulate the uptake Correspondence fo: L. Galzigna, Ph.D., M.D. Professor of Biochemistry, Department of Biological Chemistry, via Trieste 75, 35121 Padova, Italy.
148
of glutamate in isolated synaptic vesicles [ 41 and a coupling of such an uptake with the hydrolysis of ATP by a proton-pumping ATPase has been demonstrated [ 5 J . Purinergic receptors on the outer face of the cell membrane, on the other hand, seem to be quite diffused [6] and the binding of purine nucleotides is generally related with changes of ionic translocation through the membranes. Hereby we report our results on the uptake of calcium by rat synaptosomes in the presence of glutamate and with the addition of ATP. The entrance of calcium was followed, under different conditions, with an ion-selective electrode, and the hydrolysis of ATP was monitored by a 3’P-NMR technique. Materials and Methods Chemicals and instruments Ketamine (Ketalar) was obtained from Parke-Davis, Milan. All other chemicals were purchased from Sigma Chem. Co. (St. Louis, USA). Ca*+ was detected by a Ca-selective electrode by using a calomel-electrode as a reference (Metrohm AC, Herisau, CH). Synaptosomes Cerebral.synaptosomes were isolated from Wistar male rats (300-350 g body wt.) according to Nicholls [7] by using a discontinuous Ficoll density gradient. Protein was measured by the method of Lowry et al. [ 8 ] with bovine serum albumin as a standard. The synaptosomes, after separation in the gradient by centrifugation at 70,000 x g for 30 min in a Sorvall ultracentrifuge were stored in ice and all experiments were carried out within 2 h from the preparation. Ca2+ uptake assay Synaptosomal protein (0.75 mg) was preincubated with 2 mM ATP for 5 min at 23°C and then resuspended in 3 ml of a medium composed of 5 mM Tris-HCI, 4 mM MgS04, 0,25 M sucrose at a final pH of 7.4, unless otherwise stated. Some experiments were in fact carried out with different times of pre-incubation with ATP or different protein concentration. The amount of free Ca*+ detectable at different times after the addition of the synaptosomes/ATP to the medium was calculated on the basis of calibration curves carried out in the different conditions employed to transform the recorded mV values into Ca2+ concentration. ATP hydrolysis by 3’P-NMR Synaptosomal protein (2 mg) was resuspended in 2.5 ml of a medium composed of 50 mM 2-(2-hydroxy-l,l-bis(hydroxymethyl)-ethyl)-amino-ethanesulphonate, 4 mM MgS04, 0.5 mM ouabain and 0.25 M sucrose at a final pH of 7.4 with 4 mM tetraphenylphosphonium chloride as a reference. After 5 min of preincubation 5 mM ATP sodium salt neutralized with T&base was added. The resolution time, under
149
those conditions, was 1.5 min and the experiments were carried out in 10 mm tubes with a spinning rate of 10 Hz by using automated microprograms. The spectra were recorded on a Bruker MSL 300 NMR spectrometer operating at 121 MHz. Results We first checked the progressive decrease of free Ca’+, i.e. the increase of Ca*+ entry, with increasing concentration of synaptosomal protein (Fig. 1). Note that the experiments were carried out in the presence of 4 mM Mg*+ and since ATP was 2 mM there was an excess of free Mg *+ which is known to block the NMDA channel ill. In the absence of ATP no synaptosomal Ca*+ uptake was observed, while in the presence of ATP the extent of the uptake seems to increase with increasing times of preincubation of the synaptosomes with ATP (Fig. 2). Table I shows the kinetic of Ca*+ uptake expressed as mV values recorded at different times. The uptake of Ca*+ is influenced to a different extent by increasing concentrations of glutamate and NMDA (Table II). Increasing concentrations of glutamate in fact progressively stimulate the Ca2+ entry, while increasing concentrations of NMDA have, all in all, negligible effects. The amount of free Ca*+ seems to be crucial for the Ca*+ uptake
1
2 [PROTEIN] (mg)
3
Fig. 1. Effect of increasing protein concentration on the synaptosomal uptake of Ca2+. Conditions as in Materials and Methods. The preincubation time with ATP was 5 min. CaZ+ was 5 x 10” M. Means f SD. of 4 experiments.
150
\ .-C E \
f4
d E
.6 -
5
10
PREINCUBATION
15 TIME
20 (mid
Fig. 2. Effect of the times of preincubation with ATP on the uptake of Ca*+ by synaptosomes. Conditions as in Materials and Methods. Ca*+ was 5 x 10m6M. Means * SD. of 4 experiments.
process (Fig. 3); the concentration of free Ca2+ is related to the amount of bound Ca2+ by a saturation kinetic and a Scatchard’s type plot of Fig. 3 data (not shown) is linear suggesting the existence of one class of Ca2+ binding sites on the synaptosomal membrane. The action of agonists and effecters is summarized in Table III showing that the Ca2+ uptake is slightly inhibited by quisqualate while the other compounds seem uneffective. This was confirmed with higher concentrations of the effecters (not shown). The kinetics of ATP hydrolysis, followed by the disappearance of the P-ATP
TABLE I Kinetic of Ca*+ entry into synaptosomes. Ca*+ was IO-’ M and the conditions are described in Materials and Methods. Values are mean f SD. of 4 experiments. Addition (times)
mV
Medium Medium Medium Medium Medium
-5.0 -10.3 -15.0 -19.6 -23.9
+ + + + +
Ca2+ Synaptosomes/ATP Synaptosomes/ATP (1 min) Synaptosomes/ATP (2 min) SynaptosomesIATP (3 min)
mV f 0.05 f 0.08 l 0.12 f 0.11 f 0.17
-5.3 -10.0 -14.6 -18.9
TABLE II Effect of glutamate and NMDA on Ca2+ synaptosomal uptake. The values are means + S.D. from 5 experiments. Ca2+ concentration was 5 x lOm6M and a 100% value corresponds to 0.32 i: 0.08 gmol Ca2+/min/mg protein. Addition
Glutamate
NMDA
None 100 10e5 M Glutamate or NMDA 10V4M Glutamate or NMDA 5 x 10m4M Glutamate or NMDA 10V3M Glutamate or NMDA
107 135 214 200
-
-6
f f f f
5 6 8 8
91 II3 101 83
f f f f
3 5 4 5
-3 fmolll)
Fig. 3. Effect of increasing concentrations of free Ca 2c on the synaptosomal uptake of Ca*+. Conditions as in Materials and Methods. Means f SD. of 4 experiments.
152 TABLE III Synaptosomal Ca2+ uptake in the presence of effecters. The values are means f S.D. of 4 experiments; Ca2+ was 5 x low6 M and glutamate 10m4 M. A 100% value corresponds to 0.42 f 0.04 pmol Ca2+/min/mg protein. Addition
Percent
None 10m4Ketamine 10m4Quisqualate 10e4 Kainate 10e4 Glycine
100 98 80 102 93
* ze f f
2 5 6 3
signal with the 3’P-NMR technique, is faster in the presence of glutamate, Cal+ and, to a lower extent, NMDA (Table IV).
The present data are in full agreement with those obtained by using a completely different technique, i.e. the measurement of the residual radioactivity left within the synaptosomes after filtration through Millipore of a synaptosomal suspension incubated with 45Ca [ 31. Glutamate and ATP activate the uptake of Ca*+; ketamine, a dissociative anesthetic used in clinical practice, is bound to the NMDA receptorEa*+ channel and the same is true for glycine, while kainate and quisqualate are bound to other glutamate receptors. The present results suggest that the glutamate-dependent Ca*+ uptake is a rapid process since the instrumental response is immediate and linear up to 3 min from the addition of the synaptosomes. The most likely involved transport system seems to be the voltage-operated channel which is known to respond to the glutamateinduced depolarization [ 21. The usual effecters of the NMDA receptor-linked channel are in fact without any effect on the process. The ouabain-insensitive ATP hydrolysis is stimulated to a different extent by the presence of glutamate, Ca*+ and
TABLE IV ATP hydrolysis followed by the 31P-NMR technique (means f S.D. of 3 experiments) Addition
nmol ATP hydrolyzed/min/mg protein
None +4 mM Ca2+ +4 mM Glutamate +4 mM NMDA
137 287 233 160
ziz 25 zt 71 f 61 f 66
153
NMDA and this agrees with the respective effects of glutamate, ATP and NMDA on Ca2+ entry. Evidently any attempt at suggesting the clinical implications of the present findings is farfetched, although the importance of a control of the neuroexcitato~ transmission is undisputed for both normal and pathological functioning of the central nervous system. It is known that extracellular ATP can influence, at micromolar concentration, processes such as platelet aggregation, vascular tone, cardiac function, muscle contraction and, in addition, both peripheral and central neurotransmission [ 61. Nucleotides can in fact be released from neural cells by exocytosis and are metabolized by ecto-nucleotidases of which the ouabain-insensitive ATPase of this paper may be an example. The concept of ATP as a purinergic transmitter has been put forward, together with that of its neuroeffector and neuromodulator activities [ 61 but more work has to be done before these ideas can find a clinical application.
This research has been carried out with the financial help of the Ministry of Education (grant 06/89) and the Regione Veneto (grant 200/88). References 1 2 3 4 5 6 7 8
Cotman CW, Monaghan DT. Excitatory neurotransmission: NMDA receptors and Hebb-type synaptic plasticity. Ann Rev Neurosci 1988:!I:61 -80. Mayer ML, Miller RJ. Excitatory aminoacid receptors, second messengers and regulation of intracellular Ca*+ in mammalian neurons. Trends Pharmacol Sci 1990;11:254-260. Galzigna L, Bianchi M, Rizzo V. Battistin T, Scarpa M, Rigo A. ATFstimulated uptake of calcium and glutamate in rat synaptosomes. Cell B&hem. & Function 1992; in press. Naito S, Ueda T. Adenosine triphosphate-dependent uptake of glutamate into protein I-associated synaptic vesicles. J Biol Chem 1983;259:696-699. Shioi J, Naito S, Ueda T. Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane. Biochem J 1989;258:499-504. Gordon JL. Extracellular ATP:effects, sources and fate. Biochem J 1986;233:309-319. Nicholls DG. Calcium transport and proton electrochemical potential gradient in mitochondria from guinea pig cerebral cortex and rat heart. Biochem J 1978;170:511-522. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275.