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Selective activation of phosphoinositide hydrolysis by a rigid analogue of glutamate
Neuroscience Letters, 109 (1990) 157 162
157
Elsevier Scientific Publishers Ireland Ltd. NSL 06610
Selective activation of phosphoinositide hydrolysis by a rigid analogue of glutamate Manisha A. Desai and P. Jeffrey Conn Department of Pharmacology, Emory University School c~fMedicine, A tlanta, GA 30322 (U.S.A.)
(Received 5 September 1989; Revised version received 25 September 1989;Accepted 25 September 1989) Keywords': Excitatory amino acid; Phosphoinositide hydrolysis: D,L-l-Amino-l,3-cyclopentanedicar-
boxylic acid: Ibotenate; Glutamate: Inositol phosphate: Hippocampus Ibotenate and trans-D,e-l-amino-l,3-cyclopentanedicarboxylic acid (trans-ACPD) are rigid analogues of glutamate. Ibotenate has been shown to activate phosphoinositide hydrolysis in rat brain slices. We now report that trans-ACPD also stimulates phosphoinositide hydrolysis but with much higher potency and efficacythan ibotenate. The pharmacological profiles, regional distributions, and developmental regulation of the responses to ibotenate and trans-ACPD are similar, suggesting that these agonists act at the same site. However, trans-ACPD is the first agonist described that is selective for this receptor relative to other excitatory amino acid receptor subtypes. In the vertebrate central nervous system, excitatory amino acids (EAAs), such as glutamate and aspartate, are the primary neurotransmitters involved in excitatory synaptic transmission. Fast synaptic responses at E A A synapses are mediated by activation o f one o f at least three distinct ligand-operated cation channels that have been classified and n a m e d on the basis o f their preference for selective agonists. These include the N-methyl-o-aspartate ( N M D A ) , kainate (KA), and quisqualate (QUIS) receptors (see refs. 6 and 1 1 for reviews). In addition, evidence suggests that at least one distinct E A A receptor subtype exists which is not an ion channel but employs phosphoinositide hydrolysis for signal transduction [13, 14, 16, 17]. Some authors have suggested that two such E A A receptor subtypes exist [16]. In brain slices, ibotenate is the most efficacious k n o w n E A A at stimulating phosphoinositide hydrolysis. This response is activated to a lesser extent by quisqualate and glutamate [13, 14]. A l t h o u g h it has not definitively been shown that this response is mediated by direct coupling o f an E A A receptor to phosphoinositide-specific phospholipase C, the response to ibotenate can be measured in brain slices incubated in buffer with no added Ca 2+ [13], and in Xenopus oocytes that have been injected with m R N A from rat brain [17]. This suggests that the response to ibotenate is not secondary to a calcium influx or to interneuronal interactions. Correspondence: P.J. Conn, Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322 (U.S.A.).
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
158 The exact physiological roles of EAA-stimulated phosphoinositide hydrolysis are not known and no physiological effects of EAAs have been attributed to activation of this response. There are reports of EAA-induced electrophysiological responses that are not mediated by KA, QUIS, or N M D A receptors and it is possible that some of these responses may be mediated by activation of phosphoinositide hydrolysis. For instance, trans-D&- 1-amino- 1,3-cyclopentanedicarboxylic acid (trans-ACPD) increases cell firing in thalamic [8] and spinal cord neurons [10] and this response is resistant to inhibition by antagonists of KA, QUIS, and N M D A receptors, kike ibotenate, ACPD is a rigid analogue of an extended configuration of glutamate [9]. If this extended configuration is important for ibotenate-induced stimulation of phosphoinositide hydrolysis, trans-ACPD may also stimulate this response. Thus, we performed a series of experiments in which we tested the hypothesis that trans-ACPD stimulates phosphoinositide hydrolysis in rat brain slices by acting at the same receptor that mediates ibotenate-stimulated phosphoinositide hydrolysis. Male Sprague- Dawley rats were killed by decapitation, and various brain regions were dissected as previously described [3, 7]. Amygdala/pyriform cortex was defined as the portion of cerebral cortex medial to the rhinal fissure, posterior to the limbic forebrain and anterior to the amygdaloid fissure. Phosphoinositide hydrolysis was measured using a modification of the method of Berridge et al. [1] as previously described [4, 5]. This method involves measurement of agonist-induced accumulation of [-~H]inositol monophosphate (InsP) (in the presence of LiCl) in brain slices that have been labelled with [3H]inositol. Protein content was determined using the method of Bradford et al. [2]. Increasing concentrations of trans-ACPD (Tocris Neuramin, Essex, U.K.) induced accumulation of increasing amounts of radioactivity in [3H]InsP in [3H]inositollabelled hippocampal slices (Fig. l). The ECs0 of this response was 60 ~M. Consistent with previous reports [13, 14], ibotenate (Cambridge Research Biochemicals)also induced a concentration-dependent increase in phosphoinositide hydrolysis with an ECs0 value of 220/IM (Fig. 1). In the experiments presented, we did not measure the effects of concentrations of ibotenate greater than 1 mM. However, previous reports indicate that this is a maximally effective concentration [13, 14], and we have found that the effects of 3.16 and 1 mM ibotenate are the same (data not shown). trans-ACPD was more potent and more efficacious than ibotenate and induced a maximal increase in [3H]InsP accumulation of 1400_+ 85% of basal as compared to a maximal increase of 500_+49% of basal with ibotenate, cis-ACPD (1 mM) also induced an increase in [3H]InsP accumulation (990_+ 59% of basal). The effects of cisand trans-ACPD were non-additive, suggesting that these isomers act at the same receptor (data not shown). Physiological data suggest that cis-ACPD is a potent N M D A receptor agonist. However, trans-ACPD is not an agonist at KA, QUIS, or N M D A receptors [8-10], suggesting that this compound may be selective for the phosphoinositide hydrolysislinked EAA receptor. We therefore tested the hypothesis that trans-ACPD acts at the same site that mediates ibotenate-stimulated phosphoinositide hydrolysis. Previous reports have shown that ibotenate-stimulated phosphoinositide hydrolysis in hip-
159 50
- A - ACPD 40
--~-
r
Ibotenate
T
T
20 -oO ' L~
10
IT"
1 1 ~ ..IF" 1/- ,t "~ ~ . . ~ _
0
....
• .....
O/
I
I
I
I
5
4.5
4
3.5
3
-Log [Agonist] (M) Fig. 1. Effect of increasing concentrations of trans-ACPD and ibotenate on accumulation of radioactivity in [3H]InsP in hippocampal slices. [3H]Inositol-labelled slices were incubated with increasing concentrations ofibotenate or trans-ACPD in the presence of 10 m M LiCI. Each test-tube contained 25 pl of gravitypacked slices (0.17+0.01 mg protein). The a m o u n t of radioactivity present in [aH]InsP at the end of a 45 min incubation is plotted on the Y-axis. These data are means of 3 separate experiments, each done in triplicate. The vertical bars represent S.E.M.
pocampal slices is not inhibited by a variety ofKA, QUIS, and NMDA receptor antagonists. However, this response can be partially blocked by L-2-amino-4-phosphonobutyric acid (L-AP4), O,L-2-amino-3-phosphonopropionate (AP3), L-serine-O-phosphate (L-SOP), or glutamate, all of which may serve as partial agonists [14, 15]. We determined the effect of these compounds on ibotenate- and trans-ACPD-stimulated phosphoinositide hydrolysis. When testing the effects of antagonists, 100 pM transACPD and 365/tM ibotenate were used. These concentrations are 1.7 times the ECs0 concentration of each agonist. We found that two EAA receptor antagonists that are ineffective at inhibiting ibotenate-induced phosphoinositide hydrolysis [13, 14], o-glutamic acid diethyl ester (1 mM) and o-glutamylglycine (1 mM), had no effect on the response to trans-ACPD (data not shown). In addition, another nonselective EAA receptor antagonist, kynurenate (1 mM; Sigma), was ineffective at inhibiting the phosphoinositide hydrolysis response to either ibotenate or trans-ACPD (Fig. 2). Consistent with previous reports [14, 15], 1 mM concentrations of L-AP4 (Tocris Neuramin), AP3 (Sigma), L-SOP (Sigma), and L-glutamic acid (Sigma) all caused some increase in [3H]InsP accumulation when added alone and partially inhibited the response to ibotenate. In addition, these compounds partially inhibited the response to trans-ACPD (Fig. 2). Furthermore, we found that 1 mM ibotenate slightly decreased the response to 100/~M trans-ACPD (ibotenate, 540___37% of basal; transACPD, 940-t-44% of basal; ibotenate+trans-ACPD, 730+37% of basal; n=3). These data are consistent with the hypothesis that ibotenate is a partial agonist at the same site that mediates the phosphoinositide hydrolysis response to trans-ACPD. However, AP3-induced inhibition of the response to trans-ACPD was not statistically significant (P=0.069). Furthermore, L-AP4 and L-SOP also inhibit norepine-
160 30
E 73 v
~BASAL
ACPD
25 ~]
Ibotenate
20
~8
o 0:
10
5
0 Control
L-AP4
AP3
L-SOP
GLU
KYN
Fig. 2. Effect of L-AP4, D,L-AP3, L-SOP, glutamate (GLU), and kynurenic acid (KYN) on basal, transACPD-stimulated, and ibotenate-stimulated accumulation of radioactivity in [3HllnsP in hippocampal slices. Basal, trans-ACPD-stimulated (100 I~M) and ibotenate-stimulated (365 I~M) formation of pH]InsP was measured as in Fig. I in the presence or absence of various additional compounds. L-AP4, D,L-AP3, L-SOP, L-glutamate, and kynurenate were added (final concentration= l mM) 15 min prior to addition of ibotenate or trans-ACPD. Incubation in the presence or absence of ibotenate or trans-ACPD continued for an additional 45 min. Each bar is the mean of 3 8 separate experiments (each done in at least triplicaret, except for the effect of kynurenate in the presence of ibotenate (2 experiments, each in triplicate) and glutamate in the presence of ibotenate (1 experiment in triplicate). The data are presented as means _+S.E.M. Student's t-tests were used to compare each value with its corresponding treatment. *P< 0.05.
p h r i n e - s t i m u l a t e d p h o s p h o i n o s i t i d e hydrolysis in h i p p o c a m p a l slices [12], suggesting that these c o m p o u n d s m a y act by a m e c h a n i s m o t h e r than c o m p e t i t i v e inhibition o f E A A receptors. Thus, these d a t a alone do not conclusively d e m o n s t r a t e that these responses are m e d i a t e d by the same E A A receptor. I b o t e n a t e - s t i m u l a t e d p h o s p h o i n o s i t i d e h y d r o l y s i s shows a d r a m a t i c decline d u r i n g p o s t n a t a l d e v e l o p m e n t [12]. F u r t h e r m o r e , there is a great deal o f regional variability in the p h o s p h o i n o s i t i d e hydrolysis response to i b o t e n a t e in rat b r a i n [12, 13]. If the responses to i b o t e n a t e a n d t r a n s - A C P D are m e d i a t e d by the same receptor, the response to t r a n s - A C P D should show a d e v e l o p m e n t a l decline similar to that o b s e r v e d with ibotenate, and the regional d i s t r i b u t i o n o f the responses to the two agonists s h o u l d be the same. Thus, we c o m p a r e d the responses to t r a n s - A C P D a n d i b o t e n a t e in several different regions o f the rat b r a i n a n d in h i p p o c a m p u s at different stages of postnatal development. Consistent with previous d a t a [12], the effect o f i b o t e n a t e on [3H]InsP a c c u m u l a tion was m u c h greater in h i p p o c a m p a l slices from 8- to 9 - d a y - o l d (1700 ___79% o f basal radioactivity; n = 4 ) a n d 15- to 16-day-old (1600-+ 54% o f basal; n = 4) rats than from 8- to 12-week-old a n i m a l s (540 + 49% o f basal; n = 3). A similar v a r i a t i o n was seen when trans-ACPD-induced a c c u m u l a t i o n o f [3H]InsP was m e a s u r e d in h i p p o c a m p a l slices f r o m 8- to 9 - d a y - o l d (2300_+ 132% o f basal radioactivity), 15- to 16-dayold (2200_+ 111% o f basal), a n d 8- to 12-week-old (1400_+ 139% o f basal) animals. In a d d i t i o n , the regional d i s t r i b u t i o n o f trans-ACPD-stimulated p h o s p h o i n o s i t i d e hydrolysis was very similar to the response to i b o t e n a t e (Fig. 3). H o w e v e r , it is interest-
161 50
.x.
m
E
o. 73
BASAL
~7~ ACPD
40
v
Ibotenate
30 >~ 20
o 10
rr 0
Hipp
OB
NC
Cer
P/M
LF
Str
AM/PC
Fig. 3. Regional distributions of trans-ACPD and ibotenate-induced accumulation of radioactivity in [3H]InsP. Formation of [3H]InsPwas measured as in Fig. 1 in slices from hippocampus (Hipp), olfactory bulb (OB), neocortex (NC), pons/medulla (P/M), limbic forebrain (LF), striatum (Str), and amygdala/ pyriform cortex (AM/PC) in the absence of added agonist, in the presence of trans-ACPD (1 mM), or in the presence of ibotenate (1 mM). Each bar is the mean +S.E.M. of 3 separate experiments each done in triplicate except for P/M which is from a single experiment done in triplicate. Student's t-tests were used to compare the effectof ibotenate with that of trans-ACPD in each brain region. *P < 0.05.
ing to note that, while t r a n s - A C P D was significantly more efficacious than ibotenate in hippocampus, pons/medulla, and striatum, there was no significant difference between the efficacies of the two agonists in other brain regions. Taken together, these data suggest that t r a n s - A C P D is a highly efficacious agonist at stimulating phosphoinositide hydrolysis in hippocampal slices and that transA C P D acts at the site that mediates the phosphoinositide hydrolysis response to ibotenate. This conclusion is based on similarities of pharmacological profiles of the responses to the two agonists, as well as similar regional distributions and developmental changes. However, these data do not rule out the possibility that t r a n s - A C P D is also acting at another receptor that is coupled to phosphoinositide hydrolysis. Recently, Monaghan et al. [I 1] cited unpublished results in which they also found that t r a n s - A C P D stimulates phosphoinositide hydrolysis. In keeping with the current EAA receptor nomenclature based on selective receptor agonists, they suggested that the phosphoinositide hydrolysis-linked EAA receptor should be named the A C P D receptor. The data reported here are consistent with this classification. None of the EAA agonists that have been shown to stimulate phosphoinositide hydrolysis previously (glutamate, ibotenate, and quisqualate) are selective for stimulating phosphoinositide hydrolysis relative to activation of other EAA receptor subtypes [6]. In contrast, evidence suggests that trans-ACPD had no effect on N M D A , Q U I S or K A receptors [10]. Thus, t r a n s - A C P D is the only selective agonist at the putative phosphoinositide hydrolysis-linked EAA receptor described to date. In addition, transA C P D is a much more efficacious agonist at stimulating phosphoinositide hydrolysis than any of the previously characterized compounds and will provide an excellent tool for determining the physiological functions of this relatively novel class of EAA receptor.
162 W e t h a n k D r s . K e n n e t h P. M i n n e m a n
a n d M a r y B. B o y l e f o r c o m m e n t s a n d s u g -
gestions regarding the manuscript. This work was supported by N,I.H. Grant BRSG S07 R R 0 5 3 6 4 a n d b y a P . M . A . F o u n d a t i o n
Research Starter Grant.
1 Berridge, M.J., Downes, P.C. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 2 Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 3 Conn, P.J. and Sanders-Bush, E., Serotonin-stimulated phosphoinositide turnover: mediation by the $2 binding site in rat cerebral cortex but not in subcortical regions, J. Pharmacol. Exp. Ther., 234 (1985) 195 203. 4 Conn, P.J. and Sanders-Bush, E., Regulation of serotonin-stimulated phosphoinositide hydrolysis: relation to the serotonin 5-HT-2 binding site, J. Neurosci., 6 (1986) 3669-3675. 5 Conn, P.J. and Wilson, K.M., Modifications to phosphoinositide signaling. In H. Wheal and J. Chad (Eds.), Cellular and Molecular Neurobiology: A Practical Approach, IRL Press, Oxford, in press. 6 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. 7 Glowinski, J. and Iversen, L.L., Regional studies ofcatecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine, and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 665-669. 8 Hall, J.G., Hicks, T.P., McLennan, H., Richardson, T.L. and Wheal, H.V., The excitation of mammalian central neurones by amino acids, J. Physiol., 286 (1979) 29 39. 9 McLennan, H., Hicks, T.P. and Liu, J.R., On the configuration of the receptors for excitatory amino acids, Neuropharmacology, 21 (1982) 549-554. l0 McLennan, H. and Liu, J.R., The action of six antagonists of the excitatory amino acids on neurones of the rat spinal cord, Exp. Brain Res., 45 (1982) 151 156. 11 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The Excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365 402. 12 Nicoletti, F., Iadarola, M.J., Wroblewski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with et-adrenoceptors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1931 -1935. 13 Nicoletti, F., Meek, J.L., ladarola, M.J., Chuang, D.M., Roth, B.L. and Costa, E., Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus, J. Neurochem., 46 (1986) 40 45. 14 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. 15 Schoepp, D.D. and Johnson, B.G., Comparison of excitatory amino acid-stimulated phosphoinositide hydrolysis and N-[3H]acetylaspartylglutamate binding in rat brain: selective inhibition of phosphoinositide hydrolysis by 2-amino-3-phosphonopropionate, J. Neurochem., 53 (1989) 273 278. 16 Sladeczek, F., Recasens, M. and Bockaert, J., A new mechanism for glutamate receptor action: phosphoinositide hydrolysis, Trends Neurosci., 11 (1988) 545 549. 17 Sugiyama, H., lto, I. and Watanabe, M., Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA, Neuron, 3 (1989) 129 132.
157
Elsevier Scientific Publishers Ireland Ltd. NSL 06610
Selective activation of phosphoinositide hydrolysis by a rigid analogue of glutamate Manisha A. Desai and P. Jeffrey Conn Department of Pharmacology, Emory University School c~fMedicine, A tlanta, GA 30322 (U.S.A.)
(Received 5 September 1989; Revised version received 25 September 1989;Accepted 25 September 1989) Keywords': Excitatory amino acid; Phosphoinositide hydrolysis: D,L-l-Amino-l,3-cyclopentanedicar-
boxylic acid: Ibotenate; Glutamate: Inositol phosphate: Hippocampus Ibotenate and trans-D,e-l-amino-l,3-cyclopentanedicarboxylic acid (trans-ACPD) are rigid analogues of glutamate. Ibotenate has been shown to activate phosphoinositide hydrolysis in rat brain slices. We now report that trans-ACPD also stimulates phosphoinositide hydrolysis but with much higher potency and efficacythan ibotenate. The pharmacological profiles, regional distributions, and developmental regulation of the responses to ibotenate and trans-ACPD are similar, suggesting that these agonists act at the same site. However, trans-ACPD is the first agonist described that is selective for this receptor relative to other excitatory amino acid receptor subtypes. In the vertebrate central nervous system, excitatory amino acids (EAAs), such as glutamate and aspartate, are the primary neurotransmitters involved in excitatory synaptic transmission. Fast synaptic responses at E A A synapses are mediated by activation o f one o f at least three distinct ligand-operated cation channels that have been classified and n a m e d on the basis o f their preference for selective agonists. These include the N-methyl-o-aspartate ( N M D A ) , kainate (KA), and quisqualate (QUIS) receptors (see refs. 6 and 1 1 for reviews). In addition, evidence suggests that at least one distinct E A A receptor subtype exists which is not an ion channel but employs phosphoinositide hydrolysis for signal transduction [13, 14, 16, 17]. Some authors have suggested that two such E A A receptor subtypes exist [16]. In brain slices, ibotenate is the most efficacious k n o w n E A A at stimulating phosphoinositide hydrolysis. This response is activated to a lesser extent by quisqualate and glutamate [13, 14]. A l t h o u g h it has not definitively been shown that this response is mediated by direct coupling o f an E A A receptor to phosphoinositide-specific phospholipase C, the response to ibotenate can be measured in brain slices incubated in buffer with no added Ca 2+ [13], and in Xenopus oocytes that have been injected with m R N A from rat brain [17]. This suggests that the response to ibotenate is not secondary to a calcium influx or to interneuronal interactions. Correspondence: P.J. Conn, Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322 (U.S.A.).
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
158 The exact physiological roles of EAA-stimulated phosphoinositide hydrolysis are not known and no physiological effects of EAAs have been attributed to activation of this response. There are reports of EAA-induced electrophysiological responses that are not mediated by KA, QUIS, or N M D A receptors and it is possible that some of these responses may be mediated by activation of phosphoinositide hydrolysis. For instance, trans-D&- 1-amino- 1,3-cyclopentanedicarboxylic acid (trans-ACPD) increases cell firing in thalamic [8] and spinal cord neurons [10] and this response is resistant to inhibition by antagonists of KA, QUIS, and N M D A receptors, kike ibotenate, ACPD is a rigid analogue of an extended configuration of glutamate [9]. If this extended configuration is important for ibotenate-induced stimulation of phosphoinositide hydrolysis, trans-ACPD may also stimulate this response. Thus, we performed a series of experiments in which we tested the hypothesis that trans-ACPD stimulates phosphoinositide hydrolysis in rat brain slices by acting at the same receptor that mediates ibotenate-stimulated phosphoinositide hydrolysis. Male Sprague- Dawley rats were killed by decapitation, and various brain regions were dissected as previously described [3, 7]. Amygdala/pyriform cortex was defined as the portion of cerebral cortex medial to the rhinal fissure, posterior to the limbic forebrain and anterior to the amygdaloid fissure. Phosphoinositide hydrolysis was measured using a modification of the method of Berridge et al. [1] as previously described [4, 5]. This method involves measurement of agonist-induced accumulation of [-~H]inositol monophosphate (InsP) (in the presence of LiCl) in brain slices that have been labelled with [3H]inositol. Protein content was determined using the method of Bradford et al. [2]. Increasing concentrations of trans-ACPD (Tocris Neuramin, Essex, U.K.) induced accumulation of increasing amounts of radioactivity in [3H]InsP in [3H]inositollabelled hippocampal slices (Fig. l). The ECs0 of this response was 60 ~M. Consistent with previous reports [13, 14], ibotenate (Cambridge Research Biochemicals)also induced a concentration-dependent increase in phosphoinositide hydrolysis with an ECs0 value of 220/IM (Fig. 1). In the experiments presented, we did not measure the effects of concentrations of ibotenate greater than 1 mM. However, previous reports indicate that this is a maximally effective concentration [13, 14], and we have found that the effects of 3.16 and 1 mM ibotenate are the same (data not shown). trans-ACPD was more potent and more efficacious than ibotenate and induced a maximal increase in [3H]InsP accumulation of 1400_+ 85% of basal as compared to a maximal increase of 500_+49% of basal with ibotenate, cis-ACPD (1 mM) also induced an increase in [3H]InsP accumulation (990_+ 59% of basal). The effects of cisand trans-ACPD were non-additive, suggesting that these isomers act at the same receptor (data not shown). Physiological data suggest that cis-ACPD is a potent N M D A receptor agonist. However, trans-ACPD is not an agonist at KA, QUIS, or N M D A receptors [8-10], suggesting that this compound may be selective for the phosphoinositide hydrolysislinked EAA receptor. We therefore tested the hypothesis that trans-ACPD acts at the same site that mediates ibotenate-stimulated phosphoinositide hydrolysis. Previous reports have shown that ibotenate-stimulated phosphoinositide hydrolysis in hip-
159 50
- A - ACPD 40
--~-
r
Ibotenate
T
T
20 -oO ' L~
10
IT"
1 1 ~ ..IF" 1/- ,t "~ ~ . . ~ _
0
....
• .....
O/
I
I
I
I
5
4.5
4
3.5
3
-Log [Agonist] (M) Fig. 1. Effect of increasing concentrations of trans-ACPD and ibotenate on accumulation of radioactivity in [3H]InsP in hippocampal slices. [3H]Inositol-labelled slices were incubated with increasing concentrations ofibotenate or trans-ACPD in the presence of 10 m M LiCI. Each test-tube contained 25 pl of gravitypacked slices (0.17+0.01 mg protein). The a m o u n t of radioactivity present in [aH]InsP at the end of a 45 min incubation is plotted on the Y-axis. These data are means of 3 separate experiments, each done in triplicate. The vertical bars represent S.E.M.
pocampal slices is not inhibited by a variety ofKA, QUIS, and NMDA receptor antagonists. However, this response can be partially blocked by L-2-amino-4-phosphonobutyric acid (L-AP4), O,L-2-amino-3-phosphonopropionate (AP3), L-serine-O-phosphate (L-SOP), or glutamate, all of which may serve as partial agonists [14, 15]. We determined the effect of these compounds on ibotenate- and trans-ACPD-stimulated phosphoinositide hydrolysis. When testing the effects of antagonists, 100 pM transACPD and 365/tM ibotenate were used. These concentrations are 1.7 times the ECs0 concentration of each agonist. We found that two EAA receptor antagonists that are ineffective at inhibiting ibotenate-induced phosphoinositide hydrolysis [13, 14], o-glutamic acid diethyl ester (1 mM) and o-glutamylglycine (1 mM), had no effect on the response to trans-ACPD (data not shown). In addition, another nonselective EAA receptor antagonist, kynurenate (1 mM; Sigma), was ineffective at inhibiting the phosphoinositide hydrolysis response to either ibotenate or trans-ACPD (Fig. 2). Consistent with previous reports [14, 15], 1 mM concentrations of L-AP4 (Tocris Neuramin), AP3 (Sigma), L-SOP (Sigma), and L-glutamic acid (Sigma) all caused some increase in [3H]InsP accumulation when added alone and partially inhibited the response to ibotenate. In addition, these compounds partially inhibited the response to trans-ACPD (Fig. 2). Furthermore, we found that 1 mM ibotenate slightly decreased the response to 100/~M trans-ACPD (ibotenate, 540___37% of basal; transACPD, 940-t-44% of basal; ibotenate+trans-ACPD, 730+37% of basal; n=3). These data are consistent with the hypothesis that ibotenate is a partial agonist at the same site that mediates the phosphoinositide hydrolysis response to trans-ACPD. However, AP3-induced inhibition of the response to trans-ACPD was not statistically significant (P=0.069). Furthermore, L-AP4 and L-SOP also inhibit norepine-
160 30
E 73 v
~BASAL
ACPD
25 ~]
Ibotenate
20
~8
o 0:
10
5
0 Control
L-AP4
AP3
L-SOP
GLU
KYN
Fig. 2. Effect of L-AP4, D,L-AP3, L-SOP, glutamate (GLU), and kynurenic acid (KYN) on basal, transACPD-stimulated, and ibotenate-stimulated accumulation of radioactivity in [3HllnsP in hippocampal slices. Basal, trans-ACPD-stimulated (100 I~M) and ibotenate-stimulated (365 I~M) formation of pH]InsP was measured as in Fig. I in the presence or absence of various additional compounds. L-AP4, D,L-AP3, L-SOP, L-glutamate, and kynurenate were added (final concentration= l mM) 15 min prior to addition of ibotenate or trans-ACPD. Incubation in the presence or absence of ibotenate or trans-ACPD continued for an additional 45 min. Each bar is the mean of 3 8 separate experiments (each done in at least triplicaret, except for the effect of kynurenate in the presence of ibotenate (2 experiments, each in triplicate) and glutamate in the presence of ibotenate (1 experiment in triplicate). The data are presented as means _+S.E.M. Student's t-tests were used to compare each value with its corresponding treatment. *P< 0.05.
p h r i n e - s t i m u l a t e d p h o s p h o i n o s i t i d e hydrolysis in h i p p o c a m p a l slices [12], suggesting that these c o m p o u n d s m a y act by a m e c h a n i s m o t h e r than c o m p e t i t i v e inhibition o f E A A receptors. Thus, these d a t a alone do not conclusively d e m o n s t r a t e that these responses are m e d i a t e d by the same E A A receptor. I b o t e n a t e - s t i m u l a t e d p h o s p h o i n o s i t i d e h y d r o l y s i s shows a d r a m a t i c decline d u r i n g p o s t n a t a l d e v e l o p m e n t [12]. F u r t h e r m o r e , there is a great deal o f regional variability in the p h o s p h o i n o s i t i d e hydrolysis response to i b o t e n a t e in rat b r a i n [12, 13]. If the responses to i b o t e n a t e a n d t r a n s - A C P D are m e d i a t e d by the same receptor, the response to t r a n s - A C P D should show a d e v e l o p m e n t a l decline similar to that o b s e r v e d with ibotenate, and the regional d i s t r i b u t i o n o f the responses to the two agonists s h o u l d be the same. Thus, we c o m p a r e d the responses to t r a n s - A C P D a n d i b o t e n a t e in several different regions o f the rat b r a i n a n d in h i p p o c a m p u s at different stages of postnatal development. Consistent with previous d a t a [12], the effect o f i b o t e n a t e on [3H]InsP a c c u m u l a tion was m u c h greater in h i p p o c a m p a l slices from 8- to 9 - d a y - o l d (1700 ___79% o f basal radioactivity; n = 4 ) a n d 15- to 16-day-old (1600-+ 54% o f basal; n = 4) rats than from 8- to 12-week-old a n i m a l s (540 + 49% o f basal; n = 3). A similar v a r i a t i o n was seen when trans-ACPD-induced a c c u m u l a t i o n o f [3H]InsP was m e a s u r e d in h i p p o c a m p a l slices f r o m 8- to 9 - d a y - o l d (2300_+ 132% o f basal radioactivity), 15- to 16-dayold (2200_+ 111% o f basal), a n d 8- to 12-week-old (1400_+ 139% o f basal) animals. In a d d i t i o n , the regional d i s t r i b u t i o n o f trans-ACPD-stimulated p h o s p h o i n o s i t i d e hydrolysis was very similar to the response to i b o t e n a t e (Fig. 3). H o w e v e r , it is interest-
161 50
.x.
m
E
o. 73
BASAL
~7~ ACPD
40
v
Ibotenate
30 >~ 20
o 10
rr 0
Hipp
OB
NC
Cer
P/M
LF
Str
AM/PC
Fig. 3. Regional distributions of trans-ACPD and ibotenate-induced accumulation of radioactivity in [3H]InsP. Formation of [3H]InsPwas measured as in Fig. 1 in slices from hippocampus (Hipp), olfactory bulb (OB), neocortex (NC), pons/medulla (P/M), limbic forebrain (LF), striatum (Str), and amygdala/ pyriform cortex (AM/PC) in the absence of added agonist, in the presence of trans-ACPD (1 mM), or in the presence of ibotenate (1 mM). Each bar is the mean +S.E.M. of 3 separate experiments each done in triplicate except for P/M which is from a single experiment done in triplicate. Student's t-tests were used to compare the effectof ibotenate with that of trans-ACPD in each brain region. *P < 0.05.
ing to note that, while t r a n s - A C P D was significantly more efficacious than ibotenate in hippocampus, pons/medulla, and striatum, there was no significant difference between the efficacies of the two agonists in other brain regions. Taken together, these data suggest that t r a n s - A C P D is a highly efficacious agonist at stimulating phosphoinositide hydrolysis in hippocampal slices and that transA C P D acts at the site that mediates the phosphoinositide hydrolysis response to ibotenate. This conclusion is based on similarities of pharmacological profiles of the responses to the two agonists, as well as similar regional distributions and developmental changes. However, these data do not rule out the possibility that t r a n s - A C P D is also acting at another receptor that is coupled to phosphoinositide hydrolysis. Recently, Monaghan et al. [I 1] cited unpublished results in which they also found that t r a n s - A C P D stimulates phosphoinositide hydrolysis. In keeping with the current EAA receptor nomenclature based on selective receptor agonists, they suggested that the phosphoinositide hydrolysis-linked EAA receptor should be named the A C P D receptor. The data reported here are consistent with this classification. None of the EAA agonists that have been shown to stimulate phosphoinositide hydrolysis previously (glutamate, ibotenate, and quisqualate) are selective for stimulating phosphoinositide hydrolysis relative to activation of other EAA receptor subtypes [6]. In contrast, evidence suggests that trans-ACPD had no effect on N M D A , Q U I S or K A receptors [10]. Thus, t r a n s - A C P D is the only selective agonist at the putative phosphoinositide hydrolysis-linked EAA receptor described to date. In addition, transA C P D is a much more efficacious agonist at stimulating phosphoinositide hydrolysis than any of the previously characterized compounds and will provide an excellent tool for determining the physiological functions of this relatively novel class of EAA receptor.
162 W e t h a n k D r s . K e n n e t h P. M i n n e m a n
a n d M a r y B. B o y l e f o r c o m m e n t s a n d s u g -
gestions regarding the manuscript. This work was supported by N,I.H. Grant BRSG S07 R R 0 5 3 6 4 a n d b y a P . M . A . F o u n d a t i o n
Research Starter Grant.
1 Berridge, M.J., Downes, P.C. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 2 Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 3 Conn, P.J. and Sanders-Bush, E., Serotonin-stimulated phosphoinositide turnover: mediation by the $2 binding site in rat cerebral cortex but not in subcortical regions, J. Pharmacol. Exp. Ther., 234 (1985) 195 203. 4 Conn, P.J. and Sanders-Bush, E., Regulation of serotonin-stimulated phosphoinositide hydrolysis: relation to the serotonin 5-HT-2 binding site, J. Neurosci., 6 (1986) 3669-3675. 5 Conn, P.J. and Wilson, K.M., Modifications to phosphoinositide signaling. In H. Wheal and J. Chad (Eds.), Cellular and Molecular Neurobiology: A Practical Approach, IRL Press, Oxford, in press. 6 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. 7 Glowinski, J. and Iversen, L.L., Regional studies ofcatecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine, and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 665-669. 8 Hall, J.G., Hicks, T.P., McLennan, H., Richardson, T.L. and Wheal, H.V., The excitation of mammalian central neurones by amino acids, J. Physiol., 286 (1979) 29 39. 9 McLennan, H., Hicks, T.P. and Liu, J.R., On the configuration of the receptors for excitatory amino acids, Neuropharmacology, 21 (1982) 549-554. l0 McLennan, H. and Liu, J.R., The action of six antagonists of the excitatory amino acids on neurones of the rat spinal cord, Exp. Brain Res., 45 (1982) 151 156. 11 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The Excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365 402. 12 Nicoletti, F., Iadarola, M.J., Wroblewski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with et-adrenoceptors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1931 -1935. 13 Nicoletti, F., Meek, J.L., ladarola, M.J., Chuang, D.M., Roth, B.L. and Costa, E., Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus, J. Neurochem., 46 (1986) 40 45. 14 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. 15 Schoepp, D.D. and Johnson, B.G., Comparison of excitatory amino acid-stimulated phosphoinositide hydrolysis and N-[3H]acetylaspartylglutamate binding in rat brain: selective inhibition of phosphoinositide hydrolysis by 2-amino-3-phosphonopropionate, J. Neurochem., 53 (1989) 273 278. 16 Sladeczek, F., Recasens, M. and Bockaert, J., A new mechanism for glutamate receptor action: phosphoinositide hydrolysis, Trends Neurosci., 11 (1988) 545 549. 17 Sugiyama, H., lto, I. and Watanabe, M., Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA, Neuron, 3 (1989) 129 132.