European Journal of Pharmacology, 191 (1990) 231-234 Elsevier
231
E!JP 20741
Short co~~ication
lease-enhancing glycineon Anna Pittaluga and Maurizio Raiteri Istituto di Far~ocolog~~ e Fa~acog~o~~u, ~~~oer~it~de& Studi di Gemma, ViaIe Cetnbrmo 4. I6148 Genooa, italp
Received23 September 1990.accepted 9 October 1990
The release of 13H]noradrenaline (i3H]NA) in superfused rat hippocampal synaptosomes was increased concentration dependently by N-methyl-D-aspartate (NMDA). Glycine (1 pM), ineffective on its own, doubled the efficacy of NMDA with no significant change in affinity. The effects of NMDA alone and those of NMDA + glycine were antagonized by D-2-a~n~5-phosphonopentanoate (D-AE), Mg’ +, ( + )-5-methyl-tO,l l-~~ydr~5H-~be~~~d~ cycloheptd,lO-irnine hydrogen maleate (MK-801) or 7-chloro-kynurenate (7-Cl-KYNA) but not by strychnine. The data provide a direct demonstration of the existence of presynaptic NMDA receptors. NMDA (N-methyl-D-aspartate);
Noradrenaline release; Hippocampus (rat); Synaptosomes (superfused); Glycine
1. Introduction
receptors has not been demonstrated so far and therefore the possibility was considered (see for instance Greenamyre and Young, 1989) that NMDA receptors are essentially postsynaptic and located on dendrites and/or cell somata. Sites for both positive and negative modulators of the NMDA responses exist on the receptor
complex. Glycine has been defined as a positive allosteric modulator acting at a strychnine-insensitive site (Johnson and Ascher, 1987). However, direct effects of glycine have rarely been shown in functional studies. For instance, the NMDA-induced release of [3H]noradrenaline ([3H]NA) from hippocampal slices (a functional model proposed by various authors to study the NMDA receptor) was not directly affected by glycine, probably due to the endogenous amino acid already present in the receptor biophase (see Keith et al., 1989 and references therein). It is clear that the presence of unknown concentrations of endogenous glycine makes it impossible both to study adequately the proposed glycine allosteric m~ulation of NMDA responses and to verify the hypothesis, based on studies with Xenopus oocyte preparations, that glycine is obligato~ for the activation of the
Correspondence to: M. Raiteri, Istituto Farmacognosia, Univetsid degli Studi Cembrano 4, 16148 Genova. Italy.
NMDA receptor (Kleckner and Dingledine, 1988). As to the neuronal localization of NMDA receptors (only post- or also presynaptic?) the question could be approached by using a synaptosomal preparation. Moreover, as the technique available
The best known excitatory
ammo acid receptor is that selectivity activated by N-methyl-~-aspartate (NMDA). This receptor/ channel complex has a number of unique characteristics (see Foster and Fagg, 1987). One of the properties that seems to differentiate the NMDA receptor from most other neurotransmitter receptors concerns its neuronal localization. In fact, the existence of presynaptic NMDA
0014-2999/9O/SO3.50
di Farmacologia e di Genova. Viale
0 1990 Elsevier Science Publishers B.V. (Biomedical
Division)
in OUT I~~ratsry
(a thin layer of synaptosomes
s~~rf~~ up-down: Raiteri et al., 1974) allows e ~~~o~~a~of the endogenous impounds present in the receptor biophase, the problem of the glycine modulation could be properly addressed. In the present work the effects on [“H]NA release of NMDA and of a number of agents known to interact with the NMDA receptor/ anneI complex were investigated using superf~ssd rat hip~~pal s~aptosomes. The rest&s demonstrate the existence of release-regulating presynaptic NMDA receptors and shed some light on the role of glycine in NMDA receptor activation.
the corresponding ratio obtained under control conditions. A two-tailed Student’s t-test was used for compa~son of mean values. t3H]Noradrenaline (specific activity 30 Ci/mmol) was purchased from Amersham Radiochemical Centre (Buckinghamshire, U.K.). Glycine and strychnine hydro~hlo~de were obtained from Sigma (St. Louis, MO); 7-chloro-kynurenate (7-Cl-KYNA). D-2-amino-S-phosphonopentanoic acid (D-APS) and N-methyl-D-aspartic acid (NMDA) from Tocris Neuramin (Buckhurst Hill, U.K.); (+)-5-methyl-lo-ll-dihydro-S-H-dibenzo [a,d]cyclohepten-5,10-imine (MK 801) from Merck Schuchardt tHohenbrunn bei M~nchen, FRG),
3. Results Crude synaptosomes were prepared from the hippocampi of adult male Sprague-Dawley rats t2~250 g) as previously described (Raiteri et al., 1974) and the synaptosomai pellet was resuspended in a physiological medium of the following composition fmM): NaCl 125; KC1 3; CaCl, 1,2; MgSO, 1.2: NaH,PO, 1: NaHCO, 22; glucose 10 (aeration with 95% 0, and 5% CO, at 37°C); pH 7.2-7.4. After 15 tin of incubation at 37” C in the presence of 0.3 pM [“H]NA, identical aliquots of the synaptosomal suspension were distributed among a set of parallel superfusion chambers (Raiteri et al.. 1974). Superf~ion was started with standard medium and at t = 20 min, except when indicated. the standard medium was replaced with a medium from which MgSO, was omitted. Starting at t = 36 min, four 3-min fractions were collected. The synaptosomes were exposed to NMDA or to NMDA + glycine at the end of the first fraction: the ~tago~sts were added 8 min before NMDA. The amount of radioactivity released into each fraction was expressed as a percentage of the total synaptosomal tritium at the start of the fraction collected. The effects of drugs on [3H]NA release were evaluated by calculating the ratio between the percent effiux in the fraction co~esponding to the maximal effect of NMDA and the efflux in the first fraction collected. This ratio was compared to
Figure 1 shows that NMDA (in the absence of Mg’+) enhanced in a concentration-dependent manner the release of tritium from rat ~pp~arnpus synaptosomes prelabeled with [3H]NA. In agreement with previous results (Vezzani et al., 1987; Jones et al,, 1987) the radioactivity released was almost completely accounted for by unmetabolized [‘H]NA (data not shown). The maximal effect of NMDA (about 40%) was obtained with 100 pM of the drug. The EC,, of the agonist
Fig. 1. Stimulation of J’H]NA release by NMDA in presence ) or absence (A) of 1 PM glycine in superfused rat hippocampal synaptosomes. The amount of tritium release evoked by 100 PM NMDA, expressed as percent of tissue tritium was 1.82 (O.&% nci). See Materials and methods for experimental details. The data presented are means 5 S.E.M. of three to five experiments run in triplicate.
233
4. Discussion
h
t
t
t
I
_____ -----,--
,-+*+*ttt
100
UM NYDA
-+++,+++,
1 ’ 100 1
PM Clyclne 111 Slrfchn,nr liM 0.APS
‘--+--___
-
_-‘_,____
--+--
--_-_+___
--_*-
----__+__
---_-
_---___,_
---_*
_---____(
1
mM
Mg2’
1 UM MK-801 10 UM 7-Cl-KYNA 1 PM 7-C -KYNA
Fig. 2. Effects of different NMDA receptor antagonists on the release of [‘HjNA induced by 100 yM NMDA in the absence (left) or in the presence (right) of 1 gM glycine. The antagonists used did not themselves affect the basal tritium efflux (data not shown). See Materials and methods for experimental details. Means& S.E.M. of four to six experiments in triplicate are shown. * P < 0.001 vs. the respective control; * * P
231
E!JP 20741
Short co~~ication
lease-enhancing glycineon Anna Pittaluga and Maurizio Raiteri Istituto di Far~ocolog~~ e Fa~acog~o~~u, ~~~oer~it~de& Studi di Gemma, ViaIe Cetnbrmo 4. I6148 Genooa, italp
Received23 September 1990.accepted 9 October 1990
The release of 13H]noradrenaline (i3H]NA) in superfused rat hippocampal synaptosomes was increased concentration dependently by N-methyl-D-aspartate (NMDA). Glycine (1 pM), ineffective on its own, doubled the efficacy of NMDA with no significant change in affinity. The effects of NMDA alone and those of NMDA + glycine were antagonized by D-2-a~n~5-phosphonopentanoate (D-AE), Mg’ +, ( + )-5-methyl-tO,l l-~~ydr~5H-~be~~~d~ cycloheptd,lO-irnine hydrogen maleate (MK-801) or 7-chloro-kynurenate (7-Cl-KYNA) but not by strychnine. The data provide a direct demonstration of the existence of presynaptic NMDA receptors. NMDA (N-methyl-D-aspartate);
Noradrenaline release; Hippocampus (rat); Synaptosomes (superfused); Glycine
1. Introduction
receptors has not been demonstrated so far and therefore the possibility was considered (see for instance Greenamyre and Young, 1989) that NMDA receptors are essentially postsynaptic and located on dendrites and/or cell somata. Sites for both positive and negative modulators of the NMDA responses exist on the receptor
complex. Glycine has been defined as a positive allosteric modulator acting at a strychnine-insensitive site (Johnson and Ascher, 1987). However, direct effects of glycine have rarely been shown in functional studies. For instance, the NMDA-induced release of [3H]noradrenaline ([3H]NA) from hippocampal slices (a functional model proposed by various authors to study the NMDA receptor) was not directly affected by glycine, probably due to the endogenous amino acid already present in the receptor biophase (see Keith et al., 1989 and references therein). It is clear that the presence of unknown concentrations of endogenous glycine makes it impossible both to study adequately the proposed glycine allosteric m~ulation of NMDA responses and to verify the hypothesis, based on studies with Xenopus oocyte preparations, that glycine is obligato~ for the activation of the
Correspondence to: M. Raiteri, Istituto Farmacognosia, Univetsid degli Studi Cembrano 4, 16148 Genova. Italy.
NMDA receptor (Kleckner and Dingledine, 1988). As to the neuronal localization of NMDA receptors (only post- or also presynaptic?) the question could be approached by using a synaptosomal preparation. Moreover, as the technique available
The best known excitatory
ammo acid receptor is that selectivity activated by N-methyl-~-aspartate (NMDA). This receptor/ channel complex has a number of unique characteristics (see Foster and Fagg, 1987). One of the properties that seems to differentiate the NMDA receptor from most other neurotransmitter receptors concerns its neuronal localization. In fact, the existence of presynaptic NMDA
0014-2999/9O/SO3.50
di Farmacologia e di Genova. Viale
0 1990 Elsevier Science Publishers B.V. (Biomedical
Division)
in OUT I~~ratsry
(a thin layer of synaptosomes
s~~rf~~ up-down: Raiteri et al., 1974) allows e ~~~o~~a~of the endogenous impounds present in the receptor biophase, the problem of the glycine modulation could be properly addressed. In the present work the effects on [“H]NA release of NMDA and of a number of agents known to interact with the NMDA receptor/ anneI complex were investigated using superf~ssd rat hip~~pal s~aptosomes. The rest&s demonstrate the existence of release-regulating presynaptic NMDA receptors and shed some light on the role of glycine in NMDA receptor activation.
the corresponding ratio obtained under control conditions. A two-tailed Student’s t-test was used for compa~son of mean values. t3H]Noradrenaline (specific activity 30 Ci/mmol) was purchased from Amersham Radiochemical Centre (Buckinghamshire, U.K.). Glycine and strychnine hydro~hlo~de were obtained from Sigma (St. Louis, MO); 7-chloro-kynurenate (7-Cl-KYNA). D-2-amino-S-phosphonopentanoic acid (D-APS) and N-methyl-D-aspartic acid (NMDA) from Tocris Neuramin (Buckhurst Hill, U.K.); (+)-5-methyl-lo-ll-dihydro-S-H-dibenzo [a,d]cyclohepten-5,10-imine (MK 801) from Merck Schuchardt tHohenbrunn bei M~nchen, FRG),
3. Results Crude synaptosomes were prepared from the hippocampi of adult male Sprague-Dawley rats t2~250 g) as previously described (Raiteri et al., 1974) and the synaptosomai pellet was resuspended in a physiological medium of the following composition fmM): NaCl 125; KC1 3; CaCl, 1,2; MgSO, 1.2: NaH,PO, 1: NaHCO, 22; glucose 10 (aeration with 95% 0, and 5% CO, at 37°C); pH 7.2-7.4. After 15 tin of incubation at 37” C in the presence of 0.3 pM [“H]NA, identical aliquots of the synaptosomal suspension were distributed among a set of parallel superfusion chambers (Raiteri et al.. 1974). Superf~ion was started with standard medium and at t = 20 min, except when indicated. the standard medium was replaced with a medium from which MgSO, was omitted. Starting at t = 36 min, four 3-min fractions were collected. The synaptosomes were exposed to NMDA or to NMDA + glycine at the end of the first fraction: the ~tago~sts were added 8 min before NMDA. The amount of radioactivity released into each fraction was expressed as a percentage of the total synaptosomal tritium at the start of the fraction collected. The effects of drugs on [3H]NA release were evaluated by calculating the ratio between the percent effiux in the fraction co~esponding to the maximal effect of NMDA and the efflux in the first fraction collected. This ratio was compared to
Figure 1 shows that NMDA (in the absence of Mg’+) enhanced in a concentration-dependent manner the release of tritium from rat ~pp~arnpus synaptosomes prelabeled with [3H]NA. In agreement with previous results (Vezzani et al., 1987; Jones et al,, 1987) the radioactivity released was almost completely accounted for by unmetabolized [‘H]NA (data not shown). The maximal effect of NMDA (about 40%) was obtained with 100 pM of the drug. The EC,, of the agonist
Fig. 1. Stimulation of J’H]NA release by NMDA in presence ) or absence (A) of 1 PM glycine in superfused rat hippocampal synaptosomes. The amount of tritium release evoked by 100 PM NMDA, expressed as percent of tissue tritium was 1.82 (O.&% nci). See Materials and methods for experimental details. The data presented are means 5 S.E.M. of three to five experiments run in triplicate.
233
4. Discussion
h
t
t
t
I
_____ -----,--
,-+*+*ttt
100
UM NYDA
-+++,+++,
1 ’ 100 1
PM Clyclne 111 Slrfchn,nr liM 0.APS
‘--+--___
-
_-‘_,____
--+--
--_-_+___
--_*-
----__+__
---_-
_---___,_
---_*
_---____(
1
mM
Mg2’
1 UM MK-801 10 UM 7-Cl-KYNA 1 PM 7-C -KYNA
Fig. 2. Effects of different NMDA receptor antagonists on the release of [‘HjNA induced by 100 yM NMDA in the absence (left) or in the presence (right) of 1 gM glycine. The antagonists used did not themselves affect the basal tritium efflux (data not shown). See Materials and methods for experimental details. Means& S.E.M. of four to six experiments in triplicate are shown. * P < 0.001 vs. the respective control; * * P
