European Journal of Pharmacology, 185 (1990) 99-102
99
Elsevier EJP 20682 Short communication
Glutamate-induced inward current in a clonal nem'oblastoma cell line J a n B.F. V a n d e r V a l k a n d H e n k P.M. V i j v e r b e r g Research Institute of Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands
Received 8 June 1990, accepted 26 June 1990
The presence of glutamate-induced electrophysiological responses was examined in eight clonal neuroblastoma cell lines with the whole-cell voltage clamp technique. Only N2A cells responded to glutamate superfusion with a concentration-dependent, reversible inward current. Superfusion with the analogues kainic acid, quisqualic acid and N-methyl-D-aspartic acid also evoked inward currents but they had a smaller amplitude. The results indicate that N2A neuroblastoma cells could serve as an in vitro model to study the functional properties of glutamate receptors and associated ion channels. L-Glutamic acid; Neuroblastoma; Voltage clamp; Excitatory amino acids
1. Introduction
The glutamate (Glu) receptor appears to be a highly abundant amino acid receptor in the central nervous system (Mayer and Westbrook, 1987). Glu has received substantial interest as a putative neurotransmitter in the central nervous system since it was found to be associated with long-term potentiation, a process related to learning and memory (Collingridge and Bliss, 1987). In addition, Glu receptors appear to be involved in neurodegenerative changes induced by Glu and Glu-like excitotoxins (McGeer and McGeer, 1988). Glu is a non-selective agonist and acts at three distinct types of receptors known as kainic acid (KA)-, quisqualic acid (QA)- and N-methyl-Daspartic acid (NMDA)-preferring receptors (Watkins and Evans, 1981). These receptors are distinguished by the relative potencies of non-endogenous agonists and antagonists. So far, Glu-induced electrophysiological responses in the mammalian nervous system have
Correspondence to: J.B.F. Van der Valk, Research Institute of Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
been studied mainly with intact tissue, slice preparations or primary cultured neurones. Clonal neuroblastoma cells can be maintained in culture for prolonged periods and, upon differentiation, they express m a n y properties of mature neurones. Recently, we have used cells of the mouse neuroblastoma clone NIE-115 to study serotonin 5-HT 3 (Neijt et al., 1989) as well as neuronal nicotinic (Oortgiesen and Vijverberg, 1989) receptor-mediated ion currents. In excitatory amino acid research, neuroblastoma cell lines have only been used to study Glu binding (Malouf et al., 1984) and excitotoxicity (Prasad et al., 1980; Murphy et al., 1988). The function of Glu binding sites in N18-RE-105 neuroblastoma hybrid cells remains unknown as they appear not to be coupled to ion channels (Berry et al., 1988). The objective of the present study was to examine Glu-induced electrophysiological responses in various clonal cell-lines.
2. Materials and methods.
Rat neuroblastoma cell lines B35 and B50, mouse C1300 neuroblastoma cell lines N2A and NIE-115, and the h u m a n retinoblastoma cell line
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
100 Y79 were grown according to procedures described previously for NIE-115 cells (Oortgiesen and Vijverberg, 1989). The hybrid cell lines NG108-115, F l l and N18-RE-105 were grown similarly but with 100 /LM hypoxanthine, 0 . 4 / t M aminopterine and 16/~M thymidine added to the culture medium. Membrane currents were measured with the whole-cell patch clamp technique. Fire-polished patch pipettes had an internal diameter of 1.5-3 /xm and a resistance of 2-5 MI2. During the experiments, the membrane potential was clamped at - 8 0 mV and series resistance was compensated for 55-70%. The pipette solution contained (in mM): 75 CsC1, 75 CsF, 10 EGTA, 10 HEPES, 10 sucrose (pH 7.2 with CsOH, osmolality 310 mOsm). The external solution contained (in mM): 125 NaC1, 5.5 KC1, 1.8 CaC12, 5 glucose, 10 Na-HEPES, 10 HEPES, 53.7 sucrose (pH 7.4, osmolality 340 mOsm). Cells were continuously superfused through a capillary of 1 mm diameter positioned approximately 50-100 # m from the cell. By means of a valve system, the superfusate could be changed quickly for one containing known concentrations of L-Glu, KA, QA or NMDA. Experiments were performed at a room temperature of 20-24 ° C. The concentration-effect function i/ima x = 1 / ( 1 + (ECs0/[GIu]) n), which describes ligand-receptor interactions according to the mass-action law, was fitted to the data using a non-linear least-squares Levenberg-Marquardt algorithm.
A
5 uM
50 tiM
100 UM
j, B 1.0
f
o. t~
0.5 "o
=_ ~3
._>
0
7
6 -log EGlu]
4
3
3. Results
Fig. 1. Concentration-dependent electrophysiological effects of Glu in N2A neuroblastoma cells. (A) Superfusion with increasing concentrations of Glu for 15-s periods induced inward currents of increasing magnitude (horizontal and vertical calibration bars represent 20 s and 100 pA, respectively). (B) Concentration-effect curve of the Glu-induced inward current in 12 cells. The estimated ECs0 and slope factor of the concentrafion-effect curve were 9 + 4 /~M and 1.2+0.3, respectively. The ordinate represents the peak amplitude of the inward current normalized to the maximum current amplitude obtained with 100 #M Glu in each experiment.
Superfusion with external solution containing 50 # M Glu did not induce a detectable electrophysiological response in cells of the clones B35 (n = 9), B50 (n = 8), Y79 (n = 14), N1E-115 (n = 29), NG108-15 (n --- 7), N18-RE-105 (n = 10) and F l l (n = 10). The same concentration of Glu induced an inward current in all cells of the clone N2A (n > 50). The amplitude of the 50 # M Gluinduced response varied between cells and ranged from 60 to 500 pA. The amplitude of the inward current depended on the Glu concentration (fig. 1A). The minimum concentration of Glu to evoke a detectable inward current was 0.1 # M in most of
the cells and the maximum response was obtained with 100 /~M Glu. The ECs0 and slope factor, estimated from the concentration-effect curve in fig. 1B, were 9 + 4 / t M and 1.2 ___0.3, respectively (mean + S.D., n = 12). N2A calls were superfused with 50 /~M KA, QA or N M D A to study the sensitivity of these cells to agonists selective for sub-types of the Glu receptor. These analogues also induced inward currents, but the amplitude never exceeded 70% of that of the 50 /~M Glu-induced current (fig. 2). QA induced an inward current in all 33 cells
101 GIU
OA
KA i
NMDA
50
pAl 10s
Fig. 2. Responses to superfusion with Glu receptor agonists. Superfusion of a single N2A cell with 50 # M Glu, KA, QA and NMDA evoked inward currents of different magnitudes. In this experiment, the amplitudes of the KA=, QA- and NMDA-induced inward currents amounted to 35, 25 and 12% of the inward current induced by Glu.
tested, and the amplitude normalized to that of the Glu-induced inward current amounted to 36.8 + 13.8% (n = 14). Sensitivity to KA was apparent in 18 out of 24 cells tested. The normalized amplitude of the KA-induced inward current was 25.5 5: 9.6% (n = 8). In 17 out of 22 cells, superfusion with N M D A induced a response and the normalized amplitude amounted to 26.6 5: 12.9% (n = 13). In eight cells in which inward currents were evoked with Glu as well as with all three non-endogenous agonists, there was no significant difference in the amplitude of the inward currents induced by KA, QA and N M D A (two-tailed paired Student's t-test, 0.13 < P < 0.91).
4. Discussion The primary objective of this study was to determine whether neuroblastoma cells can be used to study functional Glu receptor properties. The present results demonstrate that differentiated neuroblastoma cells do not usually show an electrophysiological response to superfusion with Ghi. However, inward currents are observed in cells of the clone N2A superfused with Glu and Glu ana-
logues. Since the Glu response and the characteristics of its concentration-effect curve were similar to those of various mammalian neurones (Mayer and Westbrook, 1987), N2A cells may provide an in vitro model for the study of excitatory amino acid receptor properties and associated ion channels. A further characterization of the sensitivity of these cells to KA, QA and N M D A as well as to antagonists will be necessary to define the type of receptor involved in the Glu response in N2A cells. The present confirmation of the observation that Glu binding sites on N18-RE-105 hybrid cells are not coupled to ion channels (Berry et al., 1988) and the absence of Glu-induced electrophysiological responses in most neuroblastoma cell lines tested raise the question whether Glu binding sites are present in other neuroblastoma cell lines and what function these binding sites serve.
Acknowledgements We thank Dr. D. Schubert, The Salk Institute, San Diego for the gift of B35 and B50; Dr. M. Nirenberg, N.I.H., Bethesda, for N18-RE-105; Dr. M. Fishman, Massachusetts General Hospital, Boston, for F l l cells and Mr. P. Meyer, Hubrecht
102 Laboratory, Utrecht, The Netherlands for N2A cells. We are grateful to Ms. P. Martens for maintaining the cell cultures and Ing. A. De Groot for his technical advice.
References Berry, B.W., L.M. Boland, D.B. Hoch and R. Dingledine, 1988, L-glutamate binding site on N18-RE-105 neuroblastoma hybrid cells is not coupled to an ion channel, J. Neurochem. 51, 1176. Collingridge, G.L. and T.V.P. Bliss, 1987, NMDA receptors their role in long-term potentiation, Trends Neurosci. 10, 288. Malouf, A.T., R.L. Schnaar and J.T. Coyle, 1984, Characterization of a glutamic acid neurotransmitter binding site on neuroblastoma hybrid cells, J. Biol. Chem. 259, 2756. Mayer, M.L. and G.L. Westbrook, 1987, The physiology of excitatory amino acids in the vertebrate central nervous system, Prog. Neurobiol. 28, 197.
McGeer, E.G. and P.L. McGeer, 1988, Excitotoxins and animal 'models' of human disease, in: Recent Advances in Nervous System Toxicology, NATO ASI Series, Vol. 100, eds. C.L. Galli, L. Manzo and P.S. Spencer (Plenum Press, New York) p. 107. Murphy, T.H., A.T. Malouf, A. Sastre, R.L. Schnaar and J.T. Coyle, 1988, Calcium-dependent glutamate cytotoxicity in a neuronal cell line, Brain Res. 444, 325. Neijt, H.C., J.J. Plomp and H.P.M. Vijverberg, 1989, Kinetics of the membrane current mediated by 5-HT3 receptors in cultured mouse neuroblastoma cells, J. Physiol. 411,257. Oortgiesen, M. and H.P.M. Vijverberg, 1989, Properties of neuronal type acetylcholine receptors in voltage clamped mouse neuroblastoma cells, Neuroscience 31,169. Prasad, K.N., M. Nayak, J. Edwards-Prasad, S. Cummings and K. Pattisapu, 1980, Modification of glutamate - effects on neuroblastoma cells in culture by heavy metals, Life Sci. 27, 2251. Watkins, J.C. and R.H. Evans, 1981, Excitatory amino acid transmitters, Ann. Rev. Pharmacol. Toxicol. 21, 165.
99
Elsevier EJP 20682 Short communication
Glutamate-induced inward current in a clonal nem'oblastoma cell line J a n B.F. V a n d e r V a l k a n d H e n k P.M. V i j v e r b e r g Research Institute of Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands
Received 8 June 1990, accepted 26 June 1990
The presence of glutamate-induced electrophysiological responses was examined in eight clonal neuroblastoma cell lines with the whole-cell voltage clamp technique. Only N2A cells responded to glutamate superfusion with a concentration-dependent, reversible inward current. Superfusion with the analogues kainic acid, quisqualic acid and N-methyl-D-aspartic acid also evoked inward currents but they had a smaller amplitude. The results indicate that N2A neuroblastoma cells could serve as an in vitro model to study the functional properties of glutamate receptors and associated ion channels. L-Glutamic acid; Neuroblastoma; Voltage clamp; Excitatory amino acids
1. Introduction
The glutamate (Glu) receptor appears to be a highly abundant amino acid receptor in the central nervous system (Mayer and Westbrook, 1987). Glu has received substantial interest as a putative neurotransmitter in the central nervous system since it was found to be associated with long-term potentiation, a process related to learning and memory (Collingridge and Bliss, 1987). In addition, Glu receptors appear to be involved in neurodegenerative changes induced by Glu and Glu-like excitotoxins (McGeer and McGeer, 1988). Glu is a non-selective agonist and acts at three distinct types of receptors known as kainic acid (KA)-, quisqualic acid (QA)- and N-methyl-Daspartic acid (NMDA)-preferring receptors (Watkins and Evans, 1981). These receptors are distinguished by the relative potencies of non-endogenous agonists and antagonists. So far, Glu-induced electrophysiological responses in the mammalian nervous system have
Correspondence to: J.B.F. Van der Valk, Research Institute of Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
been studied mainly with intact tissue, slice preparations or primary cultured neurones. Clonal neuroblastoma cells can be maintained in culture for prolonged periods and, upon differentiation, they express m a n y properties of mature neurones. Recently, we have used cells of the mouse neuroblastoma clone NIE-115 to study serotonin 5-HT 3 (Neijt et al., 1989) as well as neuronal nicotinic (Oortgiesen and Vijverberg, 1989) receptor-mediated ion currents. In excitatory amino acid research, neuroblastoma cell lines have only been used to study Glu binding (Malouf et al., 1984) and excitotoxicity (Prasad et al., 1980; Murphy et al., 1988). The function of Glu binding sites in N18-RE-105 neuroblastoma hybrid cells remains unknown as they appear not to be coupled to ion channels (Berry et al., 1988). The objective of the present study was to examine Glu-induced electrophysiological responses in various clonal cell-lines.
2. Materials and methods.
Rat neuroblastoma cell lines B35 and B50, mouse C1300 neuroblastoma cell lines N2A and NIE-115, and the h u m a n retinoblastoma cell line
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
100 Y79 were grown according to procedures described previously for NIE-115 cells (Oortgiesen and Vijverberg, 1989). The hybrid cell lines NG108-115, F l l and N18-RE-105 were grown similarly but with 100 /LM hypoxanthine, 0 . 4 / t M aminopterine and 16/~M thymidine added to the culture medium. Membrane currents were measured with the whole-cell patch clamp technique. Fire-polished patch pipettes had an internal diameter of 1.5-3 /xm and a resistance of 2-5 MI2. During the experiments, the membrane potential was clamped at - 8 0 mV and series resistance was compensated for 55-70%. The pipette solution contained (in mM): 75 CsC1, 75 CsF, 10 EGTA, 10 HEPES, 10 sucrose (pH 7.2 with CsOH, osmolality 310 mOsm). The external solution contained (in mM): 125 NaC1, 5.5 KC1, 1.8 CaC12, 5 glucose, 10 Na-HEPES, 10 HEPES, 53.7 sucrose (pH 7.4, osmolality 340 mOsm). Cells were continuously superfused through a capillary of 1 mm diameter positioned approximately 50-100 # m from the cell. By means of a valve system, the superfusate could be changed quickly for one containing known concentrations of L-Glu, KA, QA or NMDA. Experiments were performed at a room temperature of 20-24 ° C. The concentration-effect function i/ima x = 1 / ( 1 + (ECs0/[GIu]) n), which describes ligand-receptor interactions according to the mass-action law, was fitted to the data using a non-linear least-squares Levenberg-Marquardt algorithm.
A
5 uM
50 tiM
100 UM
j, B 1.0
f
o. t~
0.5 "o
=_ ~3
._>
0
7
6 -log EGlu]
4
3
3. Results
Fig. 1. Concentration-dependent electrophysiological effects of Glu in N2A neuroblastoma cells. (A) Superfusion with increasing concentrations of Glu for 15-s periods induced inward currents of increasing magnitude (horizontal and vertical calibration bars represent 20 s and 100 pA, respectively). (B) Concentration-effect curve of the Glu-induced inward current in 12 cells. The estimated ECs0 and slope factor of the concentrafion-effect curve were 9 + 4 /~M and 1.2+0.3, respectively. The ordinate represents the peak amplitude of the inward current normalized to the maximum current amplitude obtained with 100 #M Glu in each experiment.
Superfusion with external solution containing 50 # M Glu did not induce a detectable electrophysiological response in cells of the clones B35 (n = 9), B50 (n = 8), Y79 (n = 14), N1E-115 (n = 29), NG108-15 (n --- 7), N18-RE-105 (n = 10) and F l l (n = 10). The same concentration of Glu induced an inward current in all cells of the clone N2A (n > 50). The amplitude of the 50 # M Gluinduced response varied between cells and ranged from 60 to 500 pA. The amplitude of the inward current depended on the Glu concentration (fig. 1A). The minimum concentration of Glu to evoke a detectable inward current was 0.1 # M in most of
the cells and the maximum response was obtained with 100 /~M Glu. The ECs0 and slope factor, estimated from the concentration-effect curve in fig. 1B, were 9 + 4 / t M and 1.2 ___0.3, respectively (mean + S.D., n = 12). N2A calls were superfused with 50 /~M KA, QA or N M D A to study the sensitivity of these cells to agonists selective for sub-types of the Glu receptor. These analogues also induced inward currents, but the amplitude never exceeded 70% of that of the 50 /~M Glu-induced current (fig. 2). QA induced an inward current in all 33 cells
101 GIU
OA
KA i
NMDA
50
pAl 10s
Fig. 2. Responses to superfusion with Glu receptor agonists. Superfusion of a single N2A cell with 50 # M Glu, KA, QA and NMDA evoked inward currents of different magnitudes. In this experiment, the amplitudes of the KA=, QA- and NMDA-induced inward currents amounted to 35, 25 and 12% of the inward current induced by Glu.
tested, and the amplitude normalized to that of the Glu-induced inward current amounted to 36.8 + 13.8% (n = 14). Sensitivity to KA was apparent in 18 out of 24 cells tested. The normalized amplitude of the KA-induced inward current was 25.5 5: 9.6% (n = 8). In 17 out of 22 cells, superfusion with N M D A induced a response and the normalized amplitude amounted to 26.6 5: 12.9% (n = 13). In eight cells in which inward currents were evoked with Glu as well as with all three non-endogenous agonists, there was no significant difference in the amplitude of the inward currents induced by KA, QA and N M D A (two-tailed paired Student's t-test, 0.13 < P < 0.91).
4. Discussion The primary objective of this study was to determine whether neuroblastoma cells can be used to study functional Glu receptor properties. The present results demonstrate that differentiated neuroblastoma cells do not usually show an electrophysiological response to superfusion with Ghi. However, inward currents are observed in cells of the clone N2A superfused with Glu and Glu ana-
logues. Since the Glu response and the characteristics of its concentration-effect curve were similar to those of various mammalian neurones (Mayer and Westbrook, 1987), N2A cells may provide an in vitro model for the study of excitatory amino acid receptor properties and associated ion channels. A further characterization of the sensitivity of these cells to KA, QA and N M D A as well as to antagonists will be necessary to define the type of receptor involved in the Glu response in N2A cells. The present confirmation of the observation that Glu binding sites on N18-RE-105 hybrid cells are not coupled to ion channels (Berry et al., 1988) and the absence of Glu-induced electrophysiological responses in most neuroblastoma cell lines tested raise the question whether Glu binding sites are present in other neuroblastoma cell lines and what function these binding sites serve.
Acknowledgements We thank Dr. D. Schubert, The Salk Institute, San Diego for the gift of B35 and B50; Dr. M. Nirenberg, N.I.H., Bethesda, for N18-RE-105; Dr. M. Fishman, Massachusetts General Hospital, Boston, for F l l cells and Mr. P. Meyer, Hubrecht
102 Laboratory, Utrecht, The Netherlands for N2A cells. We are grateful to Ms. P. Martens for maintaining the cell cultures and Ing. A. De Groot for his technical advice.
References Berry, B.W., L.M. Boland, D.B. Hoch and R. Dingledine, 1988, L-glutamate binding site on N18-RE-105 neuroblastoma hybrid cells is not coupled to an ion channel, J. Neurochem. 51, 1176. Collingridge, G.L. and T.V.P. Bliss, 1987, NMDA receptors their role in long-term potentiation, Trends Neurosci. 10, 288. Malouf, A.T., R.L. Schnaar and J.T. Coyle, 1984, Characterization of a glutamic acid neurotransmitter binding site on neuroblastoma hybrid cells, J. Biol. Chem. 259, 2756. Mayer, M.L. and G.L. Westbrook, 1987, The physiology of excitatory amino acids in the vertebrate central nervous system, Prog. Neurobiol. 28, 197.
McGeer, E.G. and P.L. McGeer, 1988, Excitotoxins and animal 'models' of human disease, in: Recent Advances in Nervous System Toxicology, NATO ASI Series, Vol. 100, eds. C.L. Galli, L. Manzo and P.S. Spencer (Plenum Press, New York) p. 107. Murphy, T.H., A.T. Malouf, A. Sastre, R.L. Schnaar and J.T. Coyle, 1988, Calcium-dependent glutamate cytotoxicity in a neuronal cell line, Brain Res. 444, 325. Neijt, H.C., J.J. Plomp and H.P.M. Vijverberg, 1989, Kinetics of the membrane current mediated by 5-HT3 receptors in cultured mouse neuroblastoma cells, J. Physiol. 411,257. Oortgiesen, M. and H.P.M. Vijverberg, 1989, Properties of neuronal type acetylcholine receptors in voltage clamped mouse neuroblastoma cells, Neuroscience 31,169. Prasad, K.N., M. Nayak, J. Edwards-Prasad, S. Cummings and K. Pattisapu, 1980, Modification of glutamate - effects on neuroblastoma cells in culture by heavy metals, Life Sci. 27, 2251. Watkins, J.C. and R.H. Evans, 1981, Excitatory amino acid transmitters, Ann. Rev. Pharmacol. Toxicol. 21, 165.
