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Ibotenic acid stimulates d -[3H]aspartate release from cultured cerebellar granule cells
Neuroscience Letters, 96 (1989) 345-350 Elsevier Scientific Publishers Ireland Ltd.
345
NSL 05846
Ibotenic acid stimulates D-[3H]aspartate release from cultured cerebellar granule cells J.P. H u b e r t a n d A. D o N e Centre de Recherches de Gennevilliers, Rh6ne-Poulenc Same;, Gennevilliers (France) (Received l I July 1988: Revised version received 29 September 1988; Accepted 29 September 1988) Key words.- Glutamate; Ibotenic acid: Release; Autoreceptor: Riluzole; Granule cell: Cell culture The release of D-[3H]aspartate from cultured cerebellar granule cells can be evoked by potassium depolarization or by superfusion with the exctitatory amino acids glutamic and aspartic acids. The latter phenomenon is not mediated by N-methyl-D-aspartate (NMDA)-, kainate- or quisqualate-preferring receptors. but is stimulated by ibotenic acid. Excitatory amino acid-stimulated D-[3H]aspartate release is antagonised by L-serine-O-phosphate and riluzole. These compounds did not block potassium-stimulated D-[3H]aspartale release. This pharmacology resembles an excitatory amino acid receptor coupled to inositol phosphate (PI) turnover.
Glutamic and aspartic acids are believed to produce their excitatory effects on neurones by interaction with three classes of excitatory amino acid receptor [12]. These three subtypes differ in their selectivities for the excitatory amino acids Nmethyl-D-aspartic (NMDA), quisqualic and kainic acid, in their sensitivity to antagonists, and in the ionic conductance states with which they are associated. However, certain responses to excitatory amino acids, observed in experimental approaches other than electrophysiology, are difficult to reconcile with this three receptor model [1, 11, 13]. Recently, Drejer et al. [5] have studied the pharmacology of an excitatory amino acid receptor on cultured cerebellar granule cells which regulates neurotransmitter release. This receptor does not respond to Nomethyl-D-aspartate, quisqualate or kainate, and the authors suggested that a novel fourth receptor subtype may be involved. The present study confirms and extends these results, and indicates that the receptor involved may correspond to an excitatory amino acid receptor previously shown to be coupled to phosphatidylinositide metabolism. Some of the results presented here have been published previously in abstract form
[4]. Correspondence: J.P. Hubert, Centre de Recherches de Gennevilliers, Rh6ne-Poulenc Sant& 35, Quai de Moulin-dc-Cage, 9223l Gennevilliers, France. (1304-3940'89,'$ 03.50 © 1989 Elsevier Scientitic Publishers Ireland Ltd.
346
D-[3H]aspartate was obtained from Amersham. Cell culture media and materials were from Gibco. Other chemicals were purchased from Sigma. Granule cell cultures were prepared as described by Gallo et al. [8]. Cerebella were removed from 7-day-old rat pups (Charles River) and dissociated enzymatically. The cell suspensions were plated (2.5-2.8 x i06 cells/cm 2) onto 3 cm Petri dishes in minimum essential medium supplemented with heat-inactived foetal calf serum (10%), glutamine (1 mM), glucose (20 mM), KCI (25 mM), insulin (100 mU/l) and penicillin/ streptomycin (10 mU/ml). During the second day of culture, cytosine ~-arabinoside (20/tM) was added to hinder the growth of non-neuronal cells. The cells were used between 7 and 10 days in culture. Before use, D-[3H]aspartate (1.8 x 10 7 M, 1 /tCi/dish) was added to the culture dishes for 30 min to label the transmitter pools. The cells were superfused with KrebsHCO3 buffer at 37°C at a flow rate of 2 ml/min. Non-uptaken radioactivity was eliminated with a 30 min washout period. Thenceforward, fractions (1 mt) of the superfusate were collected and counted for radioactivity. Pulses (0.5 min) ofdepolarising stimuli could be added directly to the superfusing medium before it reached the cells. Three 30 s pulses were applied at 4 min intervals and antagonists, if used, were added during the second pulse. At the end of the experiment, the radioactivity remaining in the cells was measured. The data were calculated as the percentage of the radioactivity remaining in the cells released in each fraction, and are expressed in %/min. The effects of various compounds on D-[3H]aspartate uptake was determined by adding them during the 4 min labelling period. After loading cerebellar granule cells with D-[3H]aspartate and a 30 min washout period, the tritium released into the superfusion medium fell to a steady level corresponding to a fractional release rate of 0.5%/min. Addition of L-glutamic acid (2 x 10 -~5 M) to the superfusion medium led to a rapid but reversible rise in the release of radioactivity into the superfusate. Successive pulses of L-glutamic acid evoked reproducible peaks of D-[3H]aspartate release corresponding to a quadrupling of the basal release rate. D-[3H]Aspartate release could also be evoked by increasing the potassium concentration of the superfusion medium to 55 mM (222 + 36%). Glutamic acid-evoked D-[3H]aspartate release was decreased by 72 + 13% (n = 3) when the calcium in the superfusion medium was replaced by magnesium (data not shown). Different excitatory amino acids were tested for their ability to evoke the release of D-[3H]aspartate (Fig. 1). L-Aspartic acid showed similar activity to L-glutamic acid, whilst D-glutamic acid was some hundred-fold less active. Of the 3 excitatory amino acids used to discriminate between excitatory amino acid receptor subytpes, N M D A and kainic acid were inactive and quisqualic acid produced only a small increase in D-[3H]aspartate release at millimolar concentrations. On the other hand, ibotenic acid stimulated the release of ~)-[3H]aspartate with an ECs0 of around 10 -s M. No excitatory amino acid tested inhibited o-[3H]aspartate release. The release of D-[3H]aspartate evoked by glutamic acid could be antagonised by the non-selective excitatory amino acid antagonist ~-aminoadipic acid (IC50=2.2+ 1.8 x 10 4 M), as well as by high concentrations of y-glutamylglycine (53 + 5% inhibition at I 0 3 M), L-2-aminophosphonobutyric acid (57 + 6% inhibition
347 600 -
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Fig. I. Excitatory amino acid-evoked D-[3H]aspartate release: concentration response curves for different excitatory amino acids (EAA). The data are presented as the percentage increase in o-[3H]aspartate release evoked by the EAA compared to the mean basal release in the 2 min preceding and following the pulse (i.e. I00%corresponds to no change in release), Three pulses were given to each dish, and the mean increase in release calculated. Each point represents the mean (__+S.E.M.) of the data obtained from 3 different cell preparations. The following excitatory amino acids were tested: L-glutamic acid (•), D-glutamic acid ( • ). L-aspartic acid (•), ibotenic acid (I,), quisqualic acid (C;), kainic acid ( V ) and N-methylaspartic acid ( A ).
at 10 3 M) and L-2-aminophosphonovaleric acid (52_+7% inhibition at 10 3 M, n = 3 for all compounds). The most potent c o m p o u n d s tested were serine-O-phosphate and riluzole (PK 26124), which also blocked the release o f D-[3H]aspartate evoked by ibotenic acid (Fig. 2). The activity o f serine-O-phosphate in this system resided in the t.-isomer. Neither t,-serine-O-phosphate nor riluzole were able to inhibit potassiumevoked t)-[3H]aspartate release (Fig. 3a). G l u t a m a t e - e v o k e d D-[3H]aspartate release was not modified by tetrodotoxin (Fig. 3b) nor by phencyclidine (10 6 M, data not shown). The uptake o f o-[3H]aspartate into cerebellar granule cells was not affected by ibotenic acid, b s e r i n e - O - p h o s p h a t e or riluzole at a concentration of 1 raM. o-[3H]As partate uptake was+ however, inhibited by dihydrokainic acid and by D-glutamic acid (IC50s o f l0 4 M for both c o m p o u n d s , data not shown). The present results confirm the finding o f Drejer et al. [5] that excitatory amino acids are capable o f stimulating the release o f o-[3H]aspartate from cerebellar granule cells. Like these authors, we find that the receptor responsible does not appear to correspond to any o f the three excitatory amino acid receptor subtypes defined electrophysiologically ( N M D A - , quisqualate- and kainate-preferring). This study demonstrates that this response can be elicited by ibotenic acid and blocked by the L-isomer o f serine-O-phosphate. Two different pharmacological effects o f ibotenic acid have been reported: in electrophysiological models it behaves
348
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~e
:tl ii Ill 0 -6 -5 -4 -3 Riluzole
-6 -5 -4 -3
-5 -4 -3
LSOP
DSOP
log [ A N T A G O N I S T ] (M)
Fig. 2. Inhibition of ibotenic acid-evoked D-[3H]aspartate release. Concentration-response curves for riluzole, L-serine-O-phosphate (LSOP) and o-serine-O-phosphate (DSOP). Three pulses (t0 4 M, 30 s) ofibotenic acid were used, the antagonist being added during the second. The data are expressed as the percentage of the evoked release obtained in the absence of antagonist (228 +__49%above basal for the riluzole experiments, 239_ 11% for LSOP, 250 + 8% for DSOP). Each point represents the mean + S.E.M. of the data obtained from 3 different cell preparations.
as a n a g o n i s t at N M D A - p r e f e r r i n g receptors, whilst in b r a i n slices it s t i m u l a t e s the f o r m a t i o n o f inositol p h o s p h a t e (IP) via a n o n - N M D A m e c h a n i s m sensitive to L-seri n e - O - p h o s p h a t e [3, 13, 15, 17]. T h e results r e p o r t e d in the p r e s e n t s t u d y are c o m p a t ible with the l a t t e r m e c h a n i s m . M o r e o v e r , a role for the quisqualic a c i d - p r e f e r r i n g r e c e p t o r c o u p l e d to IP f o r m a t i o n [16] can be excluded since the latter is n o t sensitive to s e r i n e - O - p h o s p h a t e [3], n o r d o e s quisqualic acid s t i m u l a t e D-[3H]aspartate release. It seems unlikely t h a t classical h e t e r o e x c h a n g e c o n t r i b u t e s to the D-[3H]aspartate release e v o k e d by ibotenic acid, since this c o m p o u n d has no m e a s u r a b l e effect on D[3H]aspartate u p t a k e at c o n c e n t r a t i o n s a h u n d r e d - f o l d higher t h a n those t h a t stimulate its release. O n the o t h e r h a n d , D-glutamic acid s t i m u l a t e s release a n d c o m p e t e s
300
.E_
zoo
100
+ RL]~-I,SOP K+ (55 raM)
+TTX +RLZ Glu (20 pM)
Fig. 3. Specificity of the antagonism of evoked o-[3H]aspartate release, a: effect of riluzole (RLZ, t0,4 M) and L-serine-O-phosphate (LSOP, I0 3 M ) on D-13H]aspartatereleaseevoked by potassium chloride (55 raM, 30 s). b: effectof riluzole(RLZ, 10 _4 M ) and tetrodotoxin (TTX, 10 -6 M ) on •-[3H]aspartate
release evoked by glutamic acid (20/xM, 30 s). The experimental protocol was the same as in Fig. 2. Each point represents the mean __+S.E.M. of the data obtained from 3 different cell preparations.
349 for r)-[3H]aspartate uptake at similar concentrations, However, the possibility that ibotenic acid m a y not act t h r o u g h a receptor-mediated process c a n n o t be ruled out. In this respect, ibotenic acid and L-serine-O-phosphate are inhibitors o f calcium/ chloride-dependent glutamate binding [7], which is t h o u g h t to be coupled to an uptake process [14]. This site is also, however, activated by quisqualic acid and differentiates poorly between the stereoisomers o f serine-O-phosphate. A n o t h e r aspect o f this response, also previously noted by Drejer et al. [5], was its sensitivity to riluzole. This c o m p o u n d showed a certain selectivity towards the excitatory amino acid response in that it did not block potassium-stimulated D-[3H]aspar tate release at concentrations which completely blocked ibotenic acid-stimulated release. On the other hand, it is possible that the inhibitory action o f riluzole is not direct, since this c o m p o u n d does not antagonise ibotenic acid-stimulated inositol phosphate formation [3]. The excitatory a m i n o acids seem to stimulate D-[3H]aspartate release by a direct action on the nerve terminal, rather than by stimulating neuronal firing, since the responses are not sensitive to tetrodotoxin. Furthermore, other excitatory amino acids which depolarise cerebellar granule cell bodies, such as N M D A [2], do not increase D-[3H]aspartate release in this model. Autoreceptors for excitatory amino acids have been studied in a n u m b e r o f other experimental models. McBean and Roberts [I 1] were the first to describe an autoreceptor that inhibits potassium-stimulated D-[3H]aspartate release in hippocampal slices, although no effects were seen on basal release. On the other hand, a kainic acid-sensitive receptor that stimulates e n d o g e n o u s glutamic acid release has been reported [6], which has indeed been described on cerebellar granule cells [9]. Finally, a receptor activated by 2 - a m i n o p h o s p h o n o b u t y r i c acid has been identified on the terminals o f the perforant path fibres in the h i p p o c a m p u s [10]. It seems therefore that the autoregulation o f glutamic acid release is extremely complex. In conclusion, this study extends the pharmacological definition o f the autoreceptot controlling transmitter release from cerebellar granule cells, and provides a functional role for the ibotenic acid-preferring receptor coupled to phospholipase C.
Collins, G.G.S., Some effects of excitatory amino acid receptor antagonists on synaptic transmission in rat olfactory cortex slices, Brain Res., 244 (1982) 311-318, 2 Cull-Candy, S.G. and Ogden, D.C., Ion channels activated by L-glutamate and GABA in cultured cerebellar neurones of the rat, Proc. R. Soc. London, Ser. B, 224 (1985) 367 373. 3 Doble, A. and Perrier, M.L., Excitatory amino acid receptors coupled to phosphatidylinositol metabolism in rat striatum, J. Neurochem., Suppl. 48 (1987) S121C. 4 Doble, A., Hubert, J.P. and Perrier, M.L., The pharmacology of excitatory amino acid receptor. Br. J. Pharmacol. Proc. Suppl., 91 (1987) 23C. 5 Drejer, J., Honore, T., Meier, E. and Schousboe, A., Pharmacologically distinct glutamate receptors on cerebellar granule cells, Life Sci., 38 (1986) 2077 2085. 6 Ferkany, J.W. and Coyle, J.T., Kainic acid selectivity stimulates the release of endogenous excitatory acidic amino acids, J. Pharmacol. Exp. Ther., 225 (1982) 399 406. 7 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103 164.
350 8 Gallo, V., Ciotti, M.T., Coletti, A., Alosi, F. and Levy, G., Selective release of glutamate from cerebellar granule ceils differentiating in culture, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 7919-7923. 9 Gallo, V., Suergiu, R., Giovannini, C. and Levi, G., Glutamate receptor subtypes in cultured cerebellar neurons: modulation of y-aminobutyric acid release, J. Neurochem., 49 (1987) 1801 1809. 10 Koerner, J.F. and Cotman, C.W, Micromolar L-2-amino-4-phosphonobutyric acid selectively inhibits perforant path synapses from the lateral entorhinal cortex, Brain Res., 216 (1981) 192 198. I I McBean, G.J. and Roberts, P.J., Glutamate-preferring receptors regulate the release of o-~H-aspartatc from rat hippocampal slices, Nature (Lond.), 291 (1981) 593 594. 12 McLennan, H.L., Receptors lbr the excitatory amino acids in the mammalian central nervous system. Prog. Neurobiol., 20 (1983) 251- 271. 13 Nicoletti, F., Wroblewski, J.T., ladarola, M.J. and Costa, E., Serine-O-phosphate, an endogenous metabolite, inhibits the stimulation of inositol phospholipid hydrolysis elicited by ibotenic acid in rat hippocampal neurons, Neuropharrnacology, 25 (1986) 335 338. 14 Pin, LP., Bockaert, J. and Recasens, M., The Ca z ~/CI- dependent L-[3H]glutamate binding: a new receptor or a particular transport process, FEBS Lett., 175 (1984) 31 36. 15 Schoepp, D.D. and Johnson, B.G., Excitatory amino acid agonist-antagonist interactions at 2-amino4-phosphonobutyric acid-sensitive quisqualate receptors coupled to phosphoinositide hydrolysis in slices of rat hippocampus, J, Neurochem., 50 (1988) 1605-1613. 16 Sugiyama, Y., Ito, I. and Hirono, C., A new type of glutamate receptor linked to inositol phospholipid metabolism, Nature (Lond.), 325 (1987) 531- 533. 17 Tyler, W., Watson, S.P. and Godfrey, P.P., Study of a novel phosphoinositide-linked excitatory aminoacid receptor sub-type in rat cerebral cortex, Br. J. Pharmacol., Proc., Suppl., 93 (1988) 152P.
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Ibotenic acid stimulates D-[3H]aspartate release from cultured cerebellar granule cells J.P. H u b e r t a n d A. D o N e Centre de Recherches de Gennevilliers, Rh6ne-Poulenc Same;, Gennevilliers (France) (Received l I July 1988: Revised version received 29 September 1988; Accepted 29 September 1988) Key words.- Glutamate; Ibotenic acid: Release; Autoreceptor: Riluzole; Granule cell: Cell culture The release of D-[3H]aspartate from cultured cerebellar granule cells can be evoked by potassium depolarization or by superfusion with the exctitatory amino acids glutamic and aspartic acids. The latter phenomenon is not mediated by N-methyl-D-aspartate (NMDA)-, kainate- or quisqualate-preferring receptors. but is stimulated by ibotenic acid. Excitatory amino acid-stimulated D-[3H]aspartate release is antagonised by L-serine-O-phosphate and riluzole. These compounds did not block potassium-stimulated D-[3H]aspartale release. This pharmacology resembles an excitatory amino acid receptor coupled to inositol phosphate (PI) turnover.
Glutamic and aspartic acids are believed to produce their excitatory effects on neurones by interaction with three classes of excitatory amino acid receptor [12]. These three subtypes differ in their selectivities for the excitatory amino acids Nmethyl-D-aspartic (NMDA), quisqualic and kainic acid, in their sensitivity to antagonists, and in the ionic conductance states with which they are associated. However, certain responses to excitatory amino acids, observed in experimental approaches other than electrophysiology, are difficult to reconcile with this three receptor model [1, 11, 13]. Recently, Drejer et al. [5] have studied the pharmacology of an excitatory amino acid receptor on cultured cerebellar granule cells which regulates neurotransmitter release. This receptor does not respond to Nomethyl-D-aspartate, quisqualate or kainate, and the authors suggested that a novel fourth receptor subtype may be involved. The present study confirms and extends these results, and indicates that the receptor involved may correspond to an excitatory amino acid receptor previously shown to be coupled to phosphatidylinositide metabolism. Some of the results presented here have been published previously in abstract form
[4]. Correspondence: J.P. Hubert, Centre de Recherches de Gennevilliers, Rh6ne-Poulenc Sant& 35, Quai de Moulin-dc-Cage, 9223l Gennevilliers, France. (1304-3940'89,'$ 03.50 © 1989 Elsevier Scientitic Publishers Ireland Ltd.
346
D-[3H]aspartate was obtained from Amersham. Cell culture media and materials were from Gibco. Other chemicals were purchased from Sigma. Granule cell cultures were prepared as described by Gallo et al. [8]. Cerebella were removed from 7-day-old rat pups (Charles River) and dissociated enzymatically. The cell suspensions were plated (2.5-2.8 x i06 cells/cm 2) onto 3 cm Petri dishes in minimum essential medium supplemented with heat-inactived foetal calf serum (10%), glutamine (1 mM), glucose (20 mM), KCI (25 mM), insulin (100 mU/l) and penicillin/ streptomycin (10 mU/ml). During the second day of culture, cytosine ~-arabinoside (20/tM) was added to hinder the growth of non-neuronal cells. The cells were used between 7 and 10 days in culture. Before use, D-[3H]aspartate (1.8 x 10 7 M, 1 /tCi/dish) was added to the culture dishes for 30 min to label the transmitter pools. The cells were superfused with KrebsHCO3 buffer at 37°C at a flow rate of 2 ml/min. Non-uptaken radioactivity was eliminated with a 30 min washout period. Thenceforward, fractions (1 mt) of the superfusate were collected and counted for radioactivity. Pulses (0.5 min) ofdepolarising stimuli could be added directly to the superfusing medium before it reached the cells. Three 30 s pulses were applied at 4 min intervals and antagonists, if used, were added during the second pulse. At the end of the experiment, the radioactivity remaining in the cells was measured. The data were calculated as the percentage of the radioactivity remaining in the cells released in each fraction, and are expressed in %/min. The effects of various compounds on D-[3H]aspartate uptake was determined by adding them during the 4 min labelling period. After loading cerebellar granule cells with D-[3H]aspartate and a 30 min washout period, the tritium released into the superfusion medium fell to a steady level corresponding to a fractional release rate of 0.5%/min. Addition of L-glutamic acid (2 x 10 -~5 M) to the superfusion medium led to a rapid but reversible rise in the release of radioactivity into the superfusate. Successive pulses of L-glutamic acid evoked reproducible peaks of D-[3H]aspartate release corresponding to a quadrupling of the basal release rate. D-[3H]Aspartate release could also be evoked by increasing the potassium concentration of the superfusion medium to 55 mM (222 + 36%). Glutamic acid-evoked D-[3H]aspartate release was decreased by 72 + 13% (n = 3) when the calcium in the superfusion medium was replaced by magnesium (data not shown). Different excitatory amino acids were tested for their ability to evoke the release of D-[3H]aspartate (Fig. 1). L-Aspartic acid showed similar activity to L-glutamic acid, whilst D-glutamic acid was some hundred-fold less active. Of the 3 excitatory amino acids used to discriminate between excitatory amino acid receptor subytpes, N M D A and kainic acid were inactive and quisqualic acid produced only a small increase in D-[3H]aspartate release at millimolar concentrations. On the other hand, ibotenic acid stimulated the release of ~)-[3H]aspartate with an ECs0 of around 10 -s M. No excitatory amino acid tested inhibited o-[3H]aspartate release. The release of D-[3H]aspartate evoked by glutamic acid could be antagonised by the non-selective excitatory amino acid antagonist ~-aminoadipic acid (IC50=2.2+ 1.8 x 10 4 M), as well as by high concentrations of y-glutamylglycine (53 + 5% inhibition at I 0 3 M), L-2-aminophosphonobutyric acid (57 + 6% inhibition
347 600 -
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300
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Fig. I. Excitatory amino acid-evoked D-[3H]aspartate release: concentration response curves for different excitatory amino acids (EAA). The data are presented as the percentage increase in o-[3H]aspartate release evoked by the EAA compared to the mean basal release in the 2 min preceding and following the pulse (i.e. I00%corresponds to no change in release), Three pulses were given to each dish, and the mean increase in release calculated. Each point represents the mean (__+S.E.M.) of the data obtained from 3 different cell preparations. The following excitatory amino acids were tested: L-glutamic acid (•), D-glutamic acid ( • ). L-aspartic acid (•), ibotenic acid (I,), quisqualic acid (C;), kainic acid ( V ) and N-methylaspartic acid ( A ).
at 10 3 M) and L-2-aminophosphonovaleric acid (52_+7% inhibition at 10 3 M, n = 3 for all compounds). The most potent c o m p o u n d s tested were serine-O-phosphate and riluzole (PK 26124), which also blocked the release o f D-[3H]aspartate evoked by ibotenic acid (Fig. 2). The activity o f serine-O-phosphate in this system resided in the t.-isomer. Neither t,-serine-O-phosphate nor riluzole were able to inhibit potassiumevoked t)-[3H]aspartate release (Fig. 3a). G l u t a m a t e - e v o k e d D-[3H]aspartate release was not modified by tetrodotoxin (Fig. 3b) nor by phencyclidine (10 6 M, data not shown). The uptake o f o-[3H]aspartate into cerebellar granule cells was not affected by ibotenic acid, b s e r i n e - O - p h o s p h a t e or riluzole at a concentration of 1 raM. o-[3H]As partate uptake was+ however, inhibited by dihydrokainic acid and by D-glutamic acid (IC50s o f l0 4 M for both c o m p o u n d s , data not shown). The present results confirm the finding o f Drejer et al. [5] that excitatory amino acids are capable o f stimulating the release o f o-[3H]aspartate from cerebellar granule cells. Like these authors, we find that the receptor responsible does not appear to correspond to any o f the three excitatory amino acid receptor subtypes defined electrophysiologically ( N M D A - , quisqualate- and kainate-preferring). This study demonstrates that this response can be elicited by ibotenic acid and blocked by the L-isomer o f serine-O-phosphate. Two different pharmacological effects o f ibotenic acid have been reported: in electrophysiological models it behaves
348
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~e
:tl ii Ill 0 -6 -5 -4 -3 Riluzole
-6 -5 -4 -3
-5 -4 -3
LSOP
DSOP
log [ A N T A G O N I S T ] (M)
Fig. 2. Inhibition of ibotenic acid-evoked D-[3H]aspartate release. Concentration-response curves for riluzole, L-serine-O-phosphate (LSOP) and o-serine-O-phosphate (DSOP). Three pulses (t0 4 M, 30 s) ofibotenic acid were used, the antagonist being added during the second. The data are expressed as the percentage of the evoked release obtained in the absence of antagonist (228 +__49%above basal for the riluzole experiments, 239_ 11% for LSOP, 250 + 8% for DSOP). Each point represents the mean + S.E.M. of the data obtained from 3 different cell preparations.
as a n a g o n i s t at N M D A - p r e f e r r i n g receptors, whilst in b r a i n slices it s t i m u l a t e s the f o r m a t i o n o f inositol p h o s p h a t e (IP) via a n o n - N M D A m e c h a n i s m sensitive to L-seri n e - O - p h o s p h a t e [3, 13, 15, 17]. T h e results r e p o r t e d in the p r e s e n t s t u d y are c o m p a t ible with the l a t t e r m e c h a n i s m . M o r e o v e r , a role for the quisqualic a c i d - p r e f e r r i n g r e c e p t o r c o u p l e d to IP f o r m a t i o n [16] can be excluded since the latter is n o t sensitive to s e r i n e - O - p h o s p h a t e [3], n o r d o e s quisqualic acid s t i m u l a t e D-[3H]aspartate release. It seems unlikely t h a t classical h e t e r o e x c h a n g e c o n t r i b u t e s to the D-[3H]aspartate release e v o k e d by ibotenic acid, since this c o m p o u n d has no m e a s u r a b l e effect on D[3H]aspartate u p t a k e at c o n c e n t r a t i o n s a h u n d r e d - f o l d higher t h a n those t h a t stimulate its release. O n the o t h e r h a n d , D-glutamic acid s t i m u l a t e s release a n d c o m p e t e s
300
.E_
zoo
100
+ RL]~-I,SOP K+ (55 raM)
+TTX +RLZ Glu (20 pM)
Fig. 3. Specificity of the antagonism of evoked o-[3H]aspartate release, a: effect of riluzole (RLZ, t0,4 M) and L-serine-O-phosphate (LSOP, I0 3 M ) on D-13H]aspartatereleaseevoked by potassium chloride (55 raM, 30 s). b: effectof riluzole(RLZ, 10 _4 M ) and tetrodotoxin (TTX, 10 -6 M ) on •-[3H]aspartate
release evoked by glutamic acid (20/xM, 30 s). The experimental protocol was the same as in Fig. 2. Each point represents the mean __+S.E.M. of the data obtained from 3 different cell preparations.
349 for r)-[3H]aspartate uptake at similar concentrations, However, the possibility that ibotenic acid m a y not act t h r o u g h a receptor-mediated process c a n n o t be ruled out. In this respect, ibotenic acid and L-serine-O-phosphate are inhibitors o f calcium/ chloride-dependent glutamate binding [7], which is t h o u g h t to be coupled to an uptake process [14]. This site is also, however, activated by quisqualic acid and differentiates poorly between the stereoisomers o f serine-O-phosphate. A n o t h e r aspect o f this response, also previously noted by Drejer et al. [5], was its sensitivity to riluzole. This c o m p o u n d showed a certain selectivity towards the excitatory amino acid response in that it did not block potassium-stimulated D-[3H]aspar tate release at concentrations which completely blocked ibotenic acid-stimulated release. On the other hand, it is possible that the inhibitory action o f riluzole is not direct, since this c o m p o u n d does not antagonise ibotenic acid-stimulated inositol phosphate formation [3]. The excitatory a m i n o acids seem to stimulate D-[3H]aspartate release by a direct action on the nerve terminal, rather than by stimulating neuronal firing, since the responses are not sensitive to tetrodotoxin. Furthermore, other excitatory amino acids which depolarise cerebellar granule cell bodies, such as N M D A [2], do not increase D-[3H]aspartate release in this model. Autoreceptors for excitatory amino acids have been studied in a n u m b e r o f other experimental models. McBean and Roberts [I 1] were the first to describe an autoreceptor that inhibits potassium-stimulated D-[3H]aspartate release in hippocampal slices, although no effects were seen on basal release. On the other hand, a kainic acid-sensitive receptor that stimulates e n d o g e n o u s glutamic acid release has been reported [6], which has indeed been described on cerebellar granule cells [9]. Finally, a receptor activated by 2 - a m i n o p h o s p h o n o b u t y r i c acid has been identified on the terminals o f the perforant path fibres in the h i p p o c a m p u s [10]. It seems therefore that the autoregulation o f glutamic acid release is extremely complex. In conclusion, this study extends the pharmacological definition o f the autoreceptot controlling transmitter release from cerebellar granule cells, and provides a functional role for the ibotenic acid-preferring receptor coupled to phospholipase C.
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