European Journal of Pharmacology, 185 (1990) 115-117
115
Elsevier EJP 0267R
Rapid communication
Presynaptic NMDA receptors stimulate noradrenaline release in the cerebral cortex Klaus Fink, Heinz Brnisch and Manfred Grthert Institute of Pharmacology and Toxicology, University of Bonn, ReuterstraBe 2b, D-5300 Bonn, F.R.G.
Received 16 July 1990, accepted 17 July 1990
Previous investigations in our laboratory revealed that N-methyl-D-aspartate (NMDA) and L-glutamate stimulate [3H]noradrenaline release in rat brain cortex slices (Fink et al., 1989; G&hert and Fink, 1989). However, the exact location of the N M D A receptor involved remained unclear. Tetrodotoxin strongly reduced the stimulatory effect of N M D A but failed to completely abolish it. This finding would be compatible with the view that at least some of the N M D A receptors are located on the nerve terminals themselves. However, this hypothesis was not supported by experiments on cortical synaptosomes, in which there was no stimulatory effect of N M D A on [3H]noradrenaline release. The experiments on synaptosomes, like those on slices, were carried out without exogenous glycine. It has become clear in recent years that activation of the glycine site, which is an important component of the N M D A receptor complex, is crucial for the excitability of the latter (Johnson and Ascher, 1987; Keith et al., 1989). Therefore, in the present study, the possibility was examined that, in superfused thin layers of synaptosomes (in contrast to slices), the amount of endogenous glycine available at the glycine recognition site might not be sufficient. In such synaptosome preparations, endogenous compounds are probably washed away so rapidly by the su-
Correspondence to: M. Grthert, Institute of Pharmacology and Toxicology,Universityof Bonn, ReuterstraSe 2b, D-5300 Bonn, F.R.G.
perfusion fluid that their concentration at corresponding recognition sites remains subthreshold. We now report data obtained in experiments on synaptosomes in which glycine was added to the superfusion fluid. Synaptosomes were prepared according to Mulder et al. (1975) from the brain cortex of male Wistar rats (200-300 g body weight). All details of the methods, including slight modifications of the preparation of synaptosomes, preincubation with [3H]noradrenaline (42.1-43.7 Ci/mmol), superfusion of the synaptosomes, composition of the superfusion fluid, calculations and statistics, have been described previously (Fink et al., 1989). At the end of the preparation procedure, 100-/~1 aliquots of the suspension of the synaptosomes were distributed on Whatman G F / B filters in the superfusion chambers. Subsequently, the synaptosomes were superfused for 60 min at a flow rate of 0.6 m l / m i n . In a first set of experiments (fig. la) NMDA, glycine or N M D A plus glycine was/were added to the superfusion fluid for 2 min after 40 min of superfusion. Whereas N M D A and glycine alone were ineffective, the combination of both produced a clear-cut stimulation of 3H overflow (which reflects [3H]noradrenaline release; see Fink et al., 1989). In the second series of experiments, L-glutamate (i.e. the endogenous agonist at N M D A receptors) was applied according to the same time schedule as described for N M D A (fig. lb). In the absence of glycine, L-glutamate weakly stimulated [3H]noradrenaline release and this ef-
0014-2999/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
116 tO, (b)
tO" (a)
I 0.5
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ch NMDA
1
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O
0
-
1
1
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-
1.3
1.3
1.3
1.3
1.3
.
.
M K 801
-
GLU
1
-
OJ
M 9 2"
TTX
f
1 1 1 I 1 mmoill (101 (101 001 0.01 0.01 m m o t / t 1.3
1.3
1.3
-
1.2
-
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.
1.3 m m o t / L -
0.1 -
-
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Fig. 1. Stimulation-evoked tritium overflow (above basal 3H efflux, not shown) from rat brain cortex synaptosomes preincubated with [3H]noradrenaline and superfused with modified Krebs solution (for composition, see Fink et al., 1989) which usually did not contain Mg 2+. Tritium overflow was stimulated by the addition of NMDA, glycine (GLY; left panel, a), or L-glutamate (GLU; right panel, b) to the superfusion fluid for 2 rnin after 40 rain of superfusion. When relevant in the experiments of panel b, glycine a n d / o r tetrodotoxin (TrX) was present from the 20th rain until the end of superfusion, and Ca 2+ was omitted from, or Mg 2+ was added to, the medium throughout superfusion. The concentrations of the drugs and relevant ions in the superfusion medium are given below the columns of panels a and b (-, absent from the medium). Means + S.E.M. of 5 (a) or 6 (b) experiments, each in quadruplicate * P < 0.05, compared to NMDA without GLY; + P < 0.05, compared to GLU without GLY; o p < 0.05 compared to GLU in the ~Hresence of GLY, but not significantly different from GLU in the absence of GLY. Absolute values for NMDA plus GLY-evoked overflow (last column of panel a) and GLU-evoked 3H overflow in the presence of GLY (second column of panel b): 0.21 +0.04 and 0.08 + 0.02 nCi, respectively.
fect was significantly increased by 10 /~mol/1 glycine. The threshold concentration of L-glutamate (in the presence of 10 /~mol/1 glycine), at which it induced a significant release was 1/~mol/1 and the maximum effect was already reached at 100 #tool/1 L-glutamate (n = 3; result not shown). Omission of Ca 2+ abolished the stimulatory effect of L-glutamate in the presence of glycine, whereas tetrodotoxin 0.3 #mol/1 did not influence this effect. Mg 2+, which blocks cation influx through
the ion channel coupled to the NMDA receptor, and ( + )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK 801), a highly potent, selective antagonist at this ion channel, reduced the L-glutamate-induced [3H]noradrenaline release (in the presence of glycine) to the same level as reached with Lglutamate alone (fig. lb). In conclusion, NMDA and L-glutamate (at a concentration of 1/~mol/1 which produced a max-
117
imal or half-maximal stimulation of [3H]noradrenaline release in cortical slices, respectively; see Fink et al., 1989) stimulated [3H]noradrenaline release from cortical synaptosomes. In the case of NMDA this release was completely, and in the case of L-glutamate it was partly dependent on exogenous glycine; presynaptic NMDA receptors probably mediate this stimulation. The glycine-independent, Mg 2÷- and MK 801-resistant component of the L-glutamate-induced [3H]noradrenaline release may be due to activation of synaptosomal kainate receptors. Why was the NMDA-evoked [3H]noradrenaline release far less pronounced in synaptosomes than in slices (0.6% compared to 10% of tissue tritium)? An explanation may be that only few NMDA receptors are located on noradrenergic varicosities. In the slice preparation with intact terminal axons and chains of varicosities (but not in individual pinched-off varicosities, i.e. synaptosomes), action potentials appear to be generated in response to NMDA receptor stimulation, and to play an important role in the release of [3H]noradrenaline. This view is consistent with the strong depressant effect of tetrodotoxin on NMDA-induced [3H]noradrenaline release in cortical slices (see above; Fink et al., 1989).
Acknowledgements We thank Mrs. H. Burisch and Mrs. I. Konrad for their skilled technical assistance and Mrs. R. Korneli for typing this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft.
References Fink, K., M. GOthert, G. Molderings and E. Schlicker, 1989, N.Methyl-D-aspartate (NMDA) receptor-mediated stimulation of noradrenaline release, but not release of other neurotransmitters, in the rat brain cortex: receptor location, characterization and desensitization, NaunynSchmiedeb. Arch. Pharmacol. 339, 514. G~thert, M. and K. Fink, 1989, Inhibition of N-methyl-Daspartate (NMDA)- and L-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol, Naunyn-Schmiedeb. Arch. Pharmacol. 340, 516. Johnson, J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature 325, 529. K¢ith, R.A., T.J. Mangano, B.A. Meiners, R.J. Stumpo, A.B. Klika, J. Patel and A.I. Salama, 1989, HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartateevoked neurotransmitter release. European J. Pharmacol. 166, 393. Mulder, A.H., W.B. Van den Berg and J.C. Stool 1975, Calcium-dependent release of radiolabeled catecholamines and serotonin from rat brain synaptosomes in a superfusion system, Brain. Res. 99, 419.
115
Elsevier EJP 0267R
Rapid communication
Presynaptic NMDA receptors stimulate noradrenaline release in the cerebral cortex Klaus Fink, Heinz Brnisch and Manfred Grthert Institute of Pharmacology and Toxicology, University of Bonn, ReuterstraBe 2b, D-5300 Bonn, F.R.G.
Received 16 July 1990, accepted 17 July 1990
Previous investigations in our laboratory revealed that N-methyl-D-aspartate (NMDA) and L-glutamate stimulate [3H]noradrenaline release in rat brain cortex slices (Fink et al., 1989; G&hert and Fink, 1989). However, the exact location of the N M D A receptor involved remained unclear. Tetrodotoxin strongly reduced the stimulatory effect of N M D A but failed to completely abolish it. This finding would be compatible with the view that at least some of the N M D A receptors are located on the nerve terminals themselves. However, this hypothesis was not supported by experiments on cortical synaptosomes, in which there was no stimulatory effect of N M D A on [3H]noradrenaline release. The experiments on synaptosomes, like those on slices, were carried out without exogenous glycine. It has become clear in recent years that activation of the glycine site, which is an important component of the N M D A receptor complex, is crucial for the excitability of the latter (Johnson and Ascher, 1987; Keith et al., 1989). Therefore, in the present study, the possibility was examined that, in superfused thin layers of synaptosomes (in contrast to slices), the amount of endogenous glycine available at the glycine recognition site might not be sufficient. In such synaptosome preparations, endogenous compounds are probably washed away so rapidly by the su-
Correspondence to: M. Grthert, Institute of Pharmacology and Toxicology,Universityof Bonn, ReuterstraSe 2b, D-5300 Bonn, F.R.G.
perfusion fluid that their concentration at corresponding recognition sites remains subthreshold. We now report data obtained in experiments on synaptosomes in which glycine was added to the superfusion fluid. Synaptosomes were prepared according to Mulder et al. (1975) from the brain cortex of male Wistar rats (200-300 g body weight). All details of the methods, including slight modifications of the preparation of synaptosomes, preincubation with [3H]noradrenaline (42.1-43.7 Ci/mmol), superfusion of the synaptosomes, composition of the superfusion fluid, calculations and statistics, have been described previously (Fink et al., 1989). At the end of the preparation procedure, 100-/~1 aliquots of the suspension of the synaptosomes were distributed on Whatman G F / B filters in the superfusion chambers. Subsequently, the synaptosomes were superfused for 60 min at a flow rate of 0.6 m l / m i n . In a first set of experiments (fig. la) NMDA, glycine or N M D A plus glycine was/were added to the superfusion fluid for 2 min after 40 min of superfusion. Whereas N M D A and glycine alone were ineffective, the combination of both produced a clear-cut stimulation of 3H overflow (which reflects [3H]noradrenaline release; see Fink et al., 1989). In the second series of experiments, L-glutamate (i.e. the endogenous agonist at N M D A receptors) was applied according to the same time schedule as described for N M D A (fig. lb). In the absence of glycine, L-glutamate weakly stimulated [3H]noradrenaline release and this ef-
0014-2999/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
116 tO, (b)
tO" (a)
I 0.5
J_
ch NMDA
1
G~ Co ~
O
0
-
1
1
0.1
-
1.3
1.3
1.3
1.3
1.3
.
.
M K 801
-
GLU
1
-
OJ
M 9 2"
TTX
f
1 1 1 I 1 mmoill (101 (101 001 0.01 0.01 m m o t / t 1.3
1.3
1.3
-
1.2
-
-
.
1.3 m m o t / L -
0.1 -
-
mmolll pmolll
0.3
pmolll
Fig. 1. Stimulation-evoked tritium overflow (above basal 3H efflux, not shown) from rat brain cortex synaptosomes preincubated with [3H]noradrenaline and superfused with modified Krebs solution (for composition, see Fink et al., 1989) which usually did not contain Mg 2+. Tritium overflow was stimulated by the addition of NMDA, glycine (GLY; left panel, a), or L-glutamate (GLU; right panel, b) to the superfusion fluid for 2 rnin after 40 rain of superfusion. When relevant in the experiments of panel b, glycine a n d / o r tetrodotoxin (TrX) was present from the 20th rain until the end of superfusion, and Ca 2+ was omitted from, or Mg 2+ was added to, the medium throughout superfusion. The concentrations of the drugs and relevant ions in the superfusion medium are given below the columns of panels a and b (-, absent from the medium). Means + S.E.M. of 5 (a) or 6 (b) experiments, each in quadruplicate * P < 0.05, compared to NMDA without GLY; + P < 0.05, compared to GLU without GLY; o p < 0.05 compared to GLU in the ~Hresence of GLY, but not significantly different from GLU in the absence of GLY. Absolute values for NMDA plus GLY-evoked overflow (last column of panel a) and GLU-evoked 3H overflow in the presence of GLY (second column of panel b): 0.21 +0.04 and 0.08 + 0.02 nCi, respectively.
fect was significantly increased by 10 /~mol/1 glycine. The threshold concentration of L-glutamate (in the presence of 10 /~mol/1 glycine), at which it induced a significant release was 1/~mol/1 and the maximum effect was already reached at 100 #tool/1 L-glutamate (n = 3; result not shown). Omission of Ca 2+ abolished the stimulatory effect of L-glutamate in the presence of glycine, whereas tetrodotoxin 0.3 #mol/1 did not influence this effect. Mg 2+, which blocks cation influx through
the ion channel coupled to the NMDA receptor, and ( + )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK 801), a highly potent, selective antagonist at this ion channel, reduced the L-glutamate-induced [3H]noradrenaline release (in the presence of glycine) to the same level as reached with Lglutamate alone (fig. lb). In conclusion, NMDA and L-glutamate (at a concentration of 1/~mol/1 which produced a max-
117
imal or half-maximal stimulation of [3H]noradrenaline release in cortical slices, respectively; see Fink et al., 1989) stimulated [3H]noradrenaline release from cortical synaptosomes. In the case of NMDA this release was completely, and in the case of L-glutamate it was partly dependent on exogenous glycine; presynaptic NMDA receptors probably mediate this stimulation. The glycine-independent, Mg 2÷- and MK 801-resistant component of the L-glutamate-induced [3H]noradrenaline release may be due to activation of synaptosomal kainate receptors. Why was the NMDA-evoked [3H]noradrenaline release far less pronounced in synaptosomes than in slices (0.6% compared to 10% of tissue tritium)? An explanation may be that only few NMDA receptors are located on noradrenergic varicosities. In the slice preparation with intact terminal axons and chains of varicosities (but not in individual pinched-off varicosities, i.e. synaptosomes), action potentials appear to be generated in response to NMDA receptor stimulation, and to play an important role in the release of [3H]noradrenaline. This view is consistent with the strong depressant effect of tetrodotoxin on NMDA-induced [3H]noradrenaline release in cortical slices (see above; Fink et al., 1989).
Acknowledgements We thank Mrs. H. Burisch and Mrs. I. Konrad for their skilled technical assistance and Mrs. R. Korneli for typing this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft.
References Fink, K., M. GOthert, G. Molderings and E. Schlicker, 1989, N.Methyl-D-aspartate (NMDA) receptor-mediated stimulation of noradrenaline release, but not release of other neurotransmitters, in the rat brain cortex: receptor location, characterization and desensitization, NaunynSchmiedeb. Arch. Pharmacol. 339, 514. G~thert, M. and K. Fink, 1989, Inhibition of N-methyl-Daspartate (NMDA)- and L-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol, Naunyn-Schmiedeb. Arch. Pharmacol. 340, 516. Johnson, J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature 325, 529. K¢ith, R.A., T.J. Mangano, B.A. Meiners, R.J. Stumpo, A.B. Klika, J. Patel and A.I. Salama, 1989, HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartateevoked neurotransmitter release. European J. Pharmacol. 166, 393. Mulder, A.H., W.B. Van den Berg and J.C. Stool 1975, Calcium-dependent release of radiolabeled catecholamines and serotonin from rat brain synaptosomes in a superfusion system, Brain. Res. 99, 419.
