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Endogenous inhibitor of GABAB and GABAA receptors
Neurochem. Int. Vol. 10, No. 1, pp. 65-70, 1987 Printed in Great Britain.All rights reserved
0197-0186/87 $3.00+ 0.00 © 1987PergamonJournals Ltd
ENDOGENOUS INHIBITOR OF GABA B AND GABA A RECEPTORS ICHIMARO YAMADA, AKIRA HIRATA, MASANOBUNAKAHIRO and HIROSHI YOSHIDA* Department of Pharmacology I, Osaka University School of Medicine,Nakanoshima, Kita-ku, Osaka 530, Japan (Received 7 April 1986; accepted 24 June 1986)
Al~tract--An endogenous inhibitor of 7-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG l-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na÷-independent [3H]GABA bindings. Scatchard plot analysis of the [3H]GABA binding to GABA "B" receptor recognition site showed this inhibitor increased the Ka value (24.1 nM to 3.6 nM) without changing the Bmax. On the other hand, Scatchard plot analysis of the [3H]GABA binding to GABA "A" receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the Kd value. These results suggest that the endogenous inhibitor functions as a modulator for GABAB and GABAA receptors.
y-Aminobutyric acid (GABA) is recognized to be a major inhibitory neurotransmitter in the mammalian central nervous system (McGeer and McGeer, 1981). Many recent biochemical and pharmacological studies have suggested that classical, bicuculline-sensitive receptors are coupled to chloride ion channels and benzodiazepine binding sites (Gavish and Snyder, 1981; Chang and Barnard, 1982; Leeb-Lungberg and Olsen, 1983; Siegel and Barnard, 1984). Furthermore, receptor binding studies have demonstrated the existence of novel GABA receptors, named GABAB sites, which are pharmacologically and anatomically distinct from bicuculline-sensitive GABA A sites (Hill and Bowery, 1981; Bowery et al., 1984; Wojcik and Neff, 1984). For instance, muscimol, isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) are potent agonists at GABA A receptors but baclofen is inactive (Bowery et al., 1984). On the other hand, GABA B receptors are stimulated selectively by baclofen and coupled negatively with adenylate cyclase (Wojcik and Neff, 1984). Earlier papers indicated the presence of an endogenous substance that inhibited [3H]GABA binding to GABA "A" receptor in synaptic membrane fractions (Toffano et al., 1978;
Yoneda and Kuriyama, 1980). Yoneda and Kuriyama reported the presence of a low molecular weight endogenous inhibitor of [3H]muscimol binding. Similarly, Toffano et al. (1978) proposed the existence of an endogenous modulator of GABA A receptors, acidic, heat-stable protein termed GABA modulin. However, no endogenous inhibitor or modulator of GABA Breceptors has yet been reported. The present paper shows the presence of an endogenous inhibitor of both GABA Band GABA Areceptors and investigations on its properties.
EXPERIMENTAL PROCEDURES
Materials [3H]GABA (30.7Ci/mmol) was purchased from New England Nuclear. (+) and (-)Baclofen were generously supplied by Ciba-Geigy, Basel. Biogel P-2 (200-400 mesh), Dowex AG 50W-X8 (H + form) and Dowex AG l-X8 (HCOO- form) were obtained from Bio-Red. All other chemicals were standard commercial products. Preparation and partial purification of endogenous inhibitor Five hundred bovine brains were used for each preparation. The striata (2 kg) were excised from fresh brains and promptly homogenized in 5 volumes of distilled and deionized water (D.D.W.). The homogenate was centrifuged at 105,000g for 15 min at 4°C and the supernatant was treated with an equal volume of n-butanol with stirring for 1 h and then centrifuged at 10,000g for 15 min at 4°C. The aqueous phase was boiled at 100°C for 10min and
*Address correspondence to: H. Yoshida, Department of Pharmacology I, Osaka University School of Medicine, Nakanoshima 4-3-57, Kita-ku, Osaka 530, Japan. 65
66
ICHII~AROYAIVIADAet
centrifuged at 24,000g for 60 min. The supernatant,was lyophilized, treated with chloroform:methanol (2:1 by"~ol) and extracted with methanol. The crude methanol extract was suspended in 100 ml of D.W.W. and applied to a 5.0 × 15.0cm Dowex AG I-X8 (HCOO- form) column previous equilibrated with D.D.W. Material was eluted with D.D.W. at a flow rate of 2.5 ml/ min and then with 1 N HCOOH. The fraction containing the inhibitor was lyophilized, dissolved in D.D.W. and applied to a Dowex AG 50W-X8 column (2.0 x 20cm, H ÷ form). The column was washed with D.D.W. and 1N pyridine and then the adsorbed inhibitor was eluted with 1 N NH4OH. Fractions with activity (1 N NH4OH fr.) were combined, lyophilized, dissolved in D.D.W. and purified by Biogel P-2 gel filtration. The elution pattern was monitored by the ninhydrin method.
Thin-layer chromatograph), Thin layer chromatography of active fraction was carried with Kieselgel G (type 60) using methanol:water (80:20) as solvent. Ten parts from origin to front of developed plate were extracted with 2 ml of D.D.W. After each extract was lyophilized and dissolved in 700/~1 of D.D.W., 100#1 of each fraction was used for determination on the inhibitory activities on specific total [3H]GABA binding.
Preparation of synaptic membranes Male Sprague-Dawley rats weighing 150--180g were decapitated and their brains except for the cerebellum were homogenized in 10 volumes of 0.32 M sucrose at 4°C in a glass homogenizer with a teflon pestle. The crude mitochondrial pellet was lysed in ice-cold D.D.W. for 30 min and centrifuged at 35,000g for 30 min and the resulting pellet was dispersed in 50 mM Tris-HCl buffer (pH 7.4) and recentrifuged at 35,000g for 30min. The final pellet was stored at - 2 0 ' C for 12-24 hr.
Binding assays ./or GABA receptors For assay of GABA A receptor binding, the frozen membranes were thawed, suspended in 50 mM Tri~HCI (pH 7.4) buffer and centrifuged at 105,000g for 15min. This procedure was repeated twice more and the final pellet was resuspended in 10volumes of 50mM Tris HC1 (pH 7.4) buffer at a concentration of approximately 1.0 mg protein/ ml. [3H]GABA binding (final concentration, 10mM) was performed at 25°C for I0 min in I ml of medium containing 50 mM Tris--HCl (pH 7.4) buffer and 10 -4 M ( - ) baclofen. Specifically bound [3H]GABA was defined as that displaced by 10-3 M GABA. For assay of GABAB receptor binding, the frozen membranes were thawed, suspended in 50 mM Tris-HCl containing 2.5raM CaCI~ and centrifuged at 105,000g for 15 min. This procedure was repeated as described above. [3H]GABA binding (final concentration 10raM) was performed at 25°C for 10 min in 1 ml of medium containing 50mM Tri~HCI (pH 7.4) buffer, 2.5mM CaC12 and 10 4M bicuculline. Specifically bound [3H]GABA was defined as that displaced by 10 3 M ( - ) baclofen. Specific total GABA-binding in purification procedures were performed according to the method for GABAB binding described above without l0 -4 M bicuculline, and nonspecific binding was taken by displacement with 10 3 M GABA.
al.
After 10min incubation for binding the mixture was filtered on a Whatman GF/F glass fiber filter under reduced pressure. The filter was washed 3 times with 2ml of incubation medium and its radioactivity was counted in a liquid scintillation counter. Three or four replicate assays were performed each time. Protein was determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin as a standard.
RESULTS
Purification of endogenous inhibitor of [3H]GABA binding The purification procedure is shown in Fig. 1 and described in detail in the Materials a n d Methods. The crude m e t h a n o l extract was c h r o m a t o g r a p h e d on an a n i o n exchange Dowex A G l-X8 column. As shown in Fig. 2, the inhibitory activity was eluted in fractions 2 to 5 with H 2 0 as eluant. T h u s the inhibitor was a basic or neutral molecule. The active fractions from Dowex A G I-X8 were then subjected to cation exchange c h r o m a t o g r a p h y o n Dowex A G 50W-X8 (Fig. 3). Three fractions with activity were eluted. The activity of the first fraction was weaker t h a n those of the o t h e r two fractions a n d e n d o g e n o u s G A B A was eluted in the second fraction with I N pyridine as eluant. The e n d o g e n o u s inhibitor in the third fraction appeared to be basic, as it was eluted with 1 N NH4OH. These results seem to indicate that the inhibitor in the third fraction is not G A B A itself. The third fraction that c o n t a i n e d the inhibitory activity was lyophilized, dissolved in D.D.W. and further applied to a Biogel P-2 gel filtration c o l u m n (Fig. 4). As shown in this figure, the inhibitory activity was eluted in low molecular weight fractions a n d the peak
Striatum of bovine brain I 5vol D.D.W. homogenization centrifugation (100,000g, 15 min) Supernatant
I n-BuOH treatment centrifugation (8,000g, 15 rain) Aqueous phase I
boiling (100°C, 10 min) centrifugation (24,000g, 60 min) Supernatant
I lyophilization CHCI3: MeOH =2:1 treatment MeOH extract Fig. 1. Procedure for partial purification of the endogenous inhibitor of [3H]GABA binding.
Endogenous inhibitor of GABAa and GABA^ receptors
67
-0-- o-- ninhydrin staining --v......-v-.-. activity 1"120
2.0
;4(
1N HCOOH ~
r..~,~r~.w..~.,v_v,.,r.~.y..,~.~..v-w..~i.v. ~¢~-~...~.~-v'-"0 "o '¢-°~ J~
0
CII
.g e-
•
•"~
O-•-~O~-O~-O-o'•'•-o~'O
~t)
O'O'O"o'o- O
2~ number
Fraction
do
1OO
Fig. 2. Elution profile of methanol extract by anion exchange chromatography on Dowex AG l-X8. The crude methanol extract (13 g) was dissolved in 10 ml of D.D.W. and applied to a 5.0 × 15.0 cm Dowex AG l-X8 column (HCOO- form). Material was eluted first with D.D.W. at a flow rate of 2.5 ml/min and then with step wise system of 1 N HCOOH. Fractions. of 100 ml were collected. Each fraction was lyophilized and resuspended in 100 ml of D.D.W., and the effects of 100 #1 aliquots on [3H]GABA binding were examined. The position of elution of the endogenous inhibitor was determined from the percentage inhibition of total [3H]GABA binding (10 nM) (V). activity was observed just after the peak of ninhydrinpositive material.
Properties and characteristics of endogenous inhibitor After gel filtration, the active fraction (fraction 4 8 4 9 ) was subjected to thin layer chromatography
(TLC) and the chromatogram was treated with ninhydrin reagent to locate amino acids and peptides. Two ninhydrin-positive spots (Rr 0.04 and 0.17) were detected, but neither had significant inhibitory activity. However, inhibitory activity on the [3H]GABA binding was found in Rf 0.5-0.6 (Fig. 5). Rf value of .....o--- o..- activity --ua-- m- n~hydrin staining
I~
H20
Jj(
2.0 " i •
jpQ-o4,
1N NH40H
1N ~ ' t ~ 94"t~'tk/
•~ j 4 1 ~-."
.. O/" ~
\
' .~-.
' 0 o-
g
o an
o -r
g
so~
1.0
.Q
_g .o
\ ~m-L r
1'0
~-.-r~-r'%.H_j
ab
do
4b
100
Fraction number
Fig. 3. Elution profile of active fractions from Dowex AG l-X8 by cation exchange chromatography on Dowex AG 50W-X8. The inhibitor enriched fractions from Dowex AG l-X8 were lyophilized, dissolved in 10 ml of D.D.W. and applied to a Dowex 50W-X8 column (2.0 × 20 cm, H + form) equilibrated with D.D.W. Material was eluted with D.D.W., 1 N pyridine and then with 1 N NH4OH. Fractions of 20 ml were collected, lyophilized and resuspended in 20 ml of D.D.W. Then 100 pl aliquots of each fraction were assayed for activity of total [3H]GABA binding. The elution position of the endogenous inhibitor was determined as described for Fig. 2. (O).
68
ICHIMARO
•
et al.
YAMADA
O activity nitdlydrin staining
-m---a-
~OOOqDOqNDOoe~e ~--~-==---
1.0
Vo
e e e o o ~ O
eeeeee
.i GABA ~W-
o~ 0.5
i i
~/~i
5O ~.~
.-%..,'" ::::::am::::::::::::::::::: 40 50 60 70 Fraction number
Pm=ml~nl~ nl,alm =m~
~" 10
20
30
100 80
90
Fig. 4. Biogel P2 chromatography of endogenous inhibitor of Na ÷-independent [3H]GABA binding. Active fractions (fr. 28-34) from the Dowex AG 50 W-X8 column were lyophilized, dissolved in 2 ml of D.D.W. and applied to a Biogel P2 column (2.4 × 105.0cm) and the material was eluted with D.D.W. at a flow rate of 0.22 ml/min. Fractions of 3.2 ml were collected and 10 #1 portions of each fraction were subjected to binding assay. (O), percent inhibition of total [3H]GABA binding; (11), absorbance at 570 nm with ninhydrin; Vo, void volume.
authentic G A B A was 0.42, but we could not find out G A B A spot on T L C of the active fraction. Accordingly, it seems reasonable to consider that the inhibitory factor is different substance(s) from G A B A . Enzymatic digestion (trypsin and carboxypeptidase Y) and boiling (at 100°C for 10 min) of this material (fraction 48-49) had no effect on its inhibitory activity. Thus the endogenous inhibitor of G A B A receptors is apparently a heat-stable and ninhydrin negative substance.
Effect of endogenous inhibitor on [3H]GABA binding The properties of the inhibitor on G A B A receptors were studied. Baclofen-sensitive GABAB sites can be detected in Tris-HC1 containing 2.5 m M CaCI 2 and 1 0 0 # M bicuculline to suppress binding to G A B A A sites. When tested under these conditions, the endogenous inhibitor inhibited [3H]GABA binding to GABAB sites with an IC50 value of 5.0 × 10 7 g/ml (Fig. 6). On the other hand, bicuculline-sensitive GABAA sites can be detected in Tris-HC1 buffer 100
.~ 1 O0
]
< 11o
< r0- i "1-
~
5o < I-1 .7t_l i
9
li!-i. ! o
i
!
i
)
1 -~ 8
Fig. 5. TLC analysis of the active fractions (fraction 48fraction 49) from Biogel P-2 gel filtration column. Spots were detected by spraying with ninhydrin reagent. Inhibitory activities on [3H]GABA binding were expressed in column. See Materials and Methods for details.
t
8
t
7 6 5 -log (inhibitor) g/ml
4
Fig. 6. Inhibition of specific [3H]GABA binding to "A" (A) and "'B" (Q) receptor recognition sites by various amounts of endogenous inhibitor. Bindings to GABAA and GABA B sites were determined as described in Materials and Methods. Synaptosomal membranes were incubated with [3H]GABA (10 nM) for 10 min at 25°C. Amount of endogenous inhibitor was expressed by dry weight of active fraction. Points are means for three experiments performed in triplicate.
Endogenous inhibitor of GABAB and GABAA receptors
effect on the Kd value and thus its inhibitory effect may be non-competitive rather than competitive. The Bmax changed from 706 fmol per mg protein to 494 fmol per mg protein in the presence of 5.0 × 10 -7 g/ml of the endogenous inhibitor (Fig. 8).
kd 24.1 nM e~o Bmax79 fmoles
.
69
DISCUSSION
Bmax77 f m o l e ~ I
0
50 Bound (fmoles /mg
i 100 protekO
Fig. 7. Scatchard plot analysis of [3H]GABA binding to "B" receptor recognition sites in the presence (A) and absence (O) of 5 x 10-7 g/ml of endogenous inhibitor. [3H]GABA was incubated at different concentrations (10-50 nM) with synaptosomal membranes (100#g protein per tube) for 10 thin at 25°C. Non-specific binding was defined as that in the presence of 10 -3 M unlabeled baclofen. For further details, see Materials and Methods. Points are means for triplicate determinations in three experiments. (Ca 2÷ free) containing 100 # M ( - ) b a c l o f e n to suppress binding to G A B A B sites. Under these conditiotls, the endogenous inhibitor inhibited [3H]GABA binding to GABAA sites with an IC50 of 2.0 x 10 -7 g/ ml (Fig. 6). Furthermore, Scatchard plot analysis of :{3H]GABA binding to "B" receptor recognition sites demonstrated that the endogenous inhibitor in~teased the dissociation constant (Kd) without changing the maximal number of binding sites (Bmax) indicating that it caused competitive inhibition. The K~ value changed from 24.1 nM to 38.6 nM in the presence of 5.0 x 10 -7 g/ml of the endogenous inhibitor (Fig. 7). On the other hand, Scatchard plot analysis of [3H]GABA binding to " A " receptor recognition sites seemed to demonstrate that the endogenous inhibitor reduced the Bmaxwithout significant
kd 155 nM
~ . = , . . . O . = ~ m Bntax 706 f moles 0
~
3 i2 1
~
0
~
--A----~A - ~ A . kd
186nM
Bmax 494 f
moles
i
i
10o
200
Bound(f moles / mg protein)
Fig. 8, Scatchard plot analysis [3H]GABA binding to "A" receptor recognition sites in the presence (A) and absence (O) of 5.0 x 10-7 g/ml of endogenous inhibitor. For details, see Fig. 6 and Materials and Methods.
Endogenous factors that modulate receptors have been found in the central nervous system (Asano and Spector, 1979; Guidotti et al., 1983; Zaczek et al., 1983; Akagawa et al., 1984; Atlas and Burstein, 1984; Rahavi et al., 1985; Diaz-Arrastia et al., 1985). We also reported the presence of a low molecular weight modulator of dopamine receptors in bovine brain striata (Hirata et al., 1983). There are also reports of the presence of endogenous modulators or inhibitors of bicuculline sensitive GABAA receptors (Toffano et al., 1978; Yoneda and Kuriyama, 1980; Kuroda et al., 1984). In this study, we demonstrated the presence of an endogenous inhibitor(s) of GABAA and GABAa receptors in bovine brain striata. This inhibitor(s) was not G A B A itself, since it was completely separated from endogenous G A B A on both Dowex 50W-X8 cation exchange and Biogel P-2 columns. Furthermore, by TLC we could not find out presence of G A B A in our inhibitor preparation and the inhibitory activity showed different location from that of GABA. The size of this endogenous substance was estimated as less than 500 Da by gel filtration on Biogel P-2. Our results also clearly indicated that the endogenous substance was distinct from the protein type of inhibitor named GABA-modulin. Bowery and his colleagues classified G A B A receptors into GABAA and GABAB classes (Hill and Bowery, 1981; Bowery et al., 1983). We examined whether the endogenous inhibitor inhibited [3H]GABA binding to GABAA or GABAB sites. We found that it inhibited the binding of [3H]GABA not only " A " receptor recognition sites but also "B" receptor recognition sites. Scatchard plot analysis of the data revealed that its inhibitory effect on [3H]GABA binding to " A " receptor recognition sites was non-competitive, whereas its inhibitory effect on [3H]GABA binding to "B" receptor recognition sites was competitive. The inhibitory action can be distinguished from the inhibition produced by G R I F (Yoneda and Kuriyama, 1980), which decreases the affinity of [3H]muscimol for GABAA receptor sites. The chemical structure of the endogenous inhibitor is unknown, but our results showed that it is a ninhydrin-negative, basic, heatstable and low molecular weight substance. This inhibitor had no significant influence on specific
70
ICHIMARO YAMADA et al.
bindings o f [3H]mepyramine, [3H]QNB and [3H]apomorphine. Accordingly, these results suggest that the e n d o g e n o u s i n h i b i t o r functions as a specific ligand for b o t h G A B A s a n d G A B A a receptors. Electrophysiological studies are n o w in progress on the physiological role of the e n d o g e n o u s inhibitor. Acknowledgements--We thank Mrs Mieko Nakamura for help in preparation of this manuscript and Mr Masamitu Sakamori (Research Laboratories, Yoshitomi Pharmaceutical Industries Ltd) for assistance in receptor binding assay. The work was supported by a Grant-in-Aid for Scientific Research form Ministry of Education, Science and Culture of Japan.
REFERENCES
Akagawa K., Hara N. and Tsukada Y. (1984) Partial purification and properties of the inhibitors of Na,KATPase and ouabain-binding in bovine central nervous system. J. Neurochem. 42, 775-780. Asano T. and Spector S. (1979) Identification of inosine and hypoxanthine as endogenous ligands for the brain benzodiazepine binding sites. Proc. Natn. Acad. Sci. U.S.A. 76, 977-981. Atlas D. and Burstein Y. (1984) Isolation of an endogenous clonidine-displacing substance from rat brain. FEBS Lett. 170, 387-390. Bowery N. G., Hill D. R. and Hudson A. L. (1983) Characteristics of GABA B receptor binding sites on rat whole brain synaptic membrane. Br. J. Pharmac. 78, 191 206. Bowery N. G., Price G. W., Hudson A. L., Hill D. R., Wilkin G. P. and Turnbull M. J. (1984) GABA receptor multiplicity. Neuropharmacology 23, 219 231. Chang L. R. and Barnard E. A. (1982) The benzodiazepine/GABA receptor complex, molecular size in the brain synaptic membrane and solution. J. Neurochem. 39, 1507 1518. Diaz-Arrastia R., Ashizawa T. and Appel S. H. (1985) Endogenous inhibitor of ligand binding to the muscarinic acetylcholine receptor. J. Neurochem. 44, 622628. Gavish M. and Snyder S. H. (1981) 7-Aminobutyric acid and benzodiazepine receptors, co-purification and characterization. Proc. Natn. Acad. Sci. U.S.A. 78, 1939- 1942.
Guidotti A., Forchetti C. M., Corda M. G., Konkel D., Bennet C. D. and Costa E. (1983) Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc. Natn. Acad. Sci. U.S.A. 80, 3531-3535. Hill P. R. and Bowery N. G. (1981) [3H]Baclofen and [3H]GABA bind to bicuculline insensitive GABA a sites in rat brain. Nature, Lond. 290, 149 152. Hirata A., Fujita N., Saito K. and Yoshida H. (1983) Endogenous regulator of dopamine receptors. Jap. J. Pharmac. Supplementum 33, 219 p. Neuroehem. Int. In Press. Kuroda H., Ogawa N., Nukina I. and Ota Z. (1984) An endogenous inhibitor of GABA receptor binding. Neurochem. Res. 9, 21 -27. Leeb-Lungberg F. and Olsen R. W. (1983) Heterogeneity of benzoidiazepine receptor interaction with 7-aminobutyric acid and barbiturate receptor sites. Molec. Pharmac. 23, 315 325. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. McGeer P. L. and McGeer E. G. (1981) Amino acid neurotransmitters. In Basic Neurochemistry, 3rd edn (Albers, R. W., Siegel G. J., Kotzman R. and Agranoff B. W.. eds), pp. 233 253. Little, Brown & Co., Boston. Rahavi M., Ventra 1. and Sarne Y. (1985) Demonstration of endogenous "imipramine-like" material in rat brain. L(fe Sci. 36, 687~593. Siegel E. and Barnard E. A. (1984) A ),-aminobutyric acid/ benzodiazepine receptor complex from bovine cerebral cortex. J. biol. Chem. 259, 7219-7223. Toffano G., Guidotti A. and Costa E. (1978) Purification of an endogenous protein inhibitor of the high affinity binding of l'-aminobutyric acid to synaptic membranes of rat brain. Proe. Natn. Acad. Sci. U.S.A. 75, 4024~4028. Wojcik W. K. and Neff N. H. (1984)),-Aminobutyric acid B receptors are negatively coupled to adenylate cyclase in brain, and in the cerebellum these receptors may be associated with granule cells. Molec. Pharmac. 25, 24-28. Yoneda Y. and Kuriyama K. (1980) Presence of low molecular weight endogenous inhibitor of 3H-muscimol binding in synaptic membranes. Nature, Lond. 285, 670 673. Zaczek R., Koller K., Cotter R., Heller D. and Coyle J. T. (1983) N-Acetylaspartylglutamate: an endogenous peptide with high affinity for a brain "glutamate" receptor. Proc. Nam. Acad. Sci. U.S.A. 80, 1116-1119.
0197-0186/87 $3.00+ 0.00 © 1987PergamonJournals Ltd
ENDOGENOUS INHIBITOR OF GABA B AND GABA A RECEPTORS ICHIMARO YAMADA, AKIRA HIRATA, MASANOBUNAKAHIRO and HIROSHI YOSHIDA* Department of Pharmacology I, Osaka University School of Medicine,Nakanoshima, Kita-ku, Osaka 530, Japan (Received 7 April 1986; accepted 24 June 1986)
Al~tract--An endogenous inhibitor of 7-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG l-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na÷-independent [3H]GABA bindings. Scatchard plot analysis of the [3H]GABA binding to GABA "B" receptor recognition site showed this inhibitor increased the Ka value (24.1 nM to 3.6 nM) without changing the Bmax. On the other hand, Scatchard plot analysis of the [3H]GABA binding to GABA "A" receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the Kd value. These results suggest that the endogenous inhibitor functions as a modulator for GABAB and GABAA receptors.
y-Aminobutyric acid (GABA) is recognized to be a major inhibitory neurotransmitter in the mammalian central nervous system (McGeer and McGeer, 1981). Many recent biochemical and pharmacological studies have suggested that classical, bicuculline-sensitive receptors are coupled to chloride ion channels and benzodiazepine binding sites (Gavish and Snyder, 1981; Chang and Barnard, 1982; Leeb-Lungberg and Olsen, 1983; Siegel and Barnard, 1984). Furthermore, receptor binding studies have demonstrated the existence of novel GABA receptors, named GABAB sites, which are pharmacologically and anatomically distinct from bicuculline-sensitive GABA A sites (Hill and Bowery, 1981; Bowery et al., 1984; Wojcik and Neff, 1984). For instance, muscimol, isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) are potent agonists at GABA A receptors but baclofen is inactive (Bowery et al., 1984). On the other hand, GABA B receptors are stimulated selectively by baclofen and coupled negatively with adenylate cyclase (Wojcik and Neff, 1984). Earlier papers indicated the presence of an endogenous substance that inhibited [3H]GABA binding to GABA "A" receptor in synaptic membrane fractions (Toffano et al., 1978;
Yoneda and Kuriyama, 1980). Yoneda and Kuriyama reported the presence of a low molecular weight endogenous inhibitor of [3H]muscimol binding. Similarly, Toffano et al. (1978) proposed the existence of an endogenous modulator of GABA A receptors, acidic, heat-stable protein termed GABA modulin. However, no endogenous inhibitor or modulator of GABA Breceptors has yet been reported. The present paper shows the presence of an endogenous inhibitor of both GABA Band GABA Areceptors and investigations on its properties.
EXPERIMENTAL PROCEDURES
Materials [3H]GABA (30.7Ci/mmol) was purchased from New England Nuclear. (+) and (-)Baclofen were generously supplied by Ciba-Geigy, Basel. Biogel P-2 (200-400 mesh), Dowex AG 50W-X8 (H + form) and Dowex AG l-X8 (HCOO- form) were obtained from Bio-Red. All other chemicals were standard commercial products. Preparation and partial purification of endogenous inhibitor Five hundred bovine brains were used for each preparation. The striata (2 kg) were excised from fresh brains and promptly homogenized in 5 volumes of distilled and deionized water (D.D.W.). The homogenate was centrifuged at 105,000g for 15 min at 4°C and the supernatant was treated with an equal volume of n-butanol with stirring for 1 h and then centrifuged at 10,000g for 15 min at 4°C. The aqueous phase was boiled at 100°C for 10min and
*Address correspondence to: H. Yoshida, Department of Pharmacology I, Osaka University School of Medicine, Nakanoshima 4-3-57, Kita-ku, Osaka 530, Japan. 65
66
ICHII~AROYAIVIADAet
centrifuged at 24,000g for 60 min. The supernatant,was lyophilized, treated with chloroform:methanol (2:1 by"~ol) and extracted with methanol. The crude methanol extract was suspended in 100 ml of D.W.W. and applied to a 5.0 × 15.0cm Dowex AG I-X8 (HCOO- form) column previous equilibrated with D.D.W. Material was eluted with D.D.W. at a flow rate of 2.5 ml/ min and then with 1 N HCOOH. The fraction containing the inhibitor was lyophilized, dissolved in D.D.W. and applied to a Dowex AG 50W-X8 column (2.0 x 20cm, H ÷ form). The column was washed with D.D.W. and 1N pyridine and then the adsorbed inhibitor was eluted with 1 N NH4OH. Fractions with activity (1 N NH4OH fr.) were combined, lyophilized, dissolved in D.D.W. and purified by Biogel P-2 gel filtration. The elution pattern was monitored by the ninhydrin method.
Thin-layer chromatograph), Thin layer chromatography of active fraction was carried with Kieselgel G (type 60) using methanol:water (80:20) as solvent. Ten parts from origin to front of developed plate were extracted with 2 ml of D.D.W. After each extract was lyophilized and dissolved in 700/~1 of D.D.W., 100#1 of each fraction was used for determination on the inhibitory activities on specific total [3H]GABA binding.
Preparation of synaptic membranes Male Sprague-Dawley rats weighing 150--180g were decapitated and their brains except for the cerebellum were homogenized in 10 volumes of 0.32 M sucrose at 4°C in a glass homogenizer with a teflon pestle. The crude mitochondrial pellet was lysed in ice-cold D.D.W. for 30 min and centrifuged at 35,000g for 30 min and the resulting pellet was dispersed in 50 mM Tris-HCl buffer (pH 7.4) and recentrifuged at 35,000g for 30min. The final pellet was stored at - 2 0 ' C for 12-24 hr.
Binding assays ./or GABA receptors For assay of GABA A receptor binding, the frozen membranes were thawed, suspended in 50 mM Tri~HCI (pH 7.4) buffer and centrifuged at 105,000g for 15min. This procedure was repeated twice more and the final pellet was resuspended in 10volumes of 50mM Tris HC1 (pH 7.4) buffer at a concentration of approximately 1.0 mg protein/ ml. [3H]GABA binding (final concentration, 10mM) was performed at 25°C for I0 min in I ml of medium containing 50 mM Tris--HCl (pH 7.4) buffer and 10 -4 M ( - ) baclofen. Specifically bound [3H]GABA was defined as that displaced by 10-3 M GABA. For assay of GABAB receptor binding, the frozen membranes were thawed, suspended in 50 mM Tris-HCl containing 2.5raM CaCI~ and centrifuged at 105,000g for 15 min. This procedure was repeated as described above. [3H]GABA binding (final concentration 10raM) was performed at 25°C for 10 min in 1 ml of medium containing 50mM Tri~HCI (pH 7.4) buffer, 2.5mM CaC12 and 10 4M bicuculline. Specifically bound [3H]GABA was defined as that displaced by 10 3 M ( - ) baclofen. Specific total GABA-binding in purification procedures were performed according to the method for GABAB binding described above without l0 -4 M bicuculline, and nonspecific binding was taken by displacement with 10 3 M GABA.
al.
After 10min incubation for binding the mixture was filtered on a Whatman GF/F glass fiber filter under reduced pressure. The filter was washed 3 times with 2ml of incubation medium and its radioactivity was counted in a liquid scintillation counter. Three or four replicate assays were performed each time. Protein was determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin as a standard.
RESULTS
Purification of endogenous inhibitor of [3H]GABA binding The purification procedure is shown in Fig. 1 and described in detail in the Materials a n d Methods. The crude m e t h a n o l extract was c h r o m a t o g r a p h e d on an a n i o n exchange Dowex A G l-X8 column. As shown in Fig. 2, the inhibitory activity was eluted in fractions 2 to 5 with H 2 0 as eluant. T h u s the inhibitor was a basic or neutral molecule. The active fractions from Dowex A G I-X8 were then subjected to cation exchange c h r o m a t o g r a p h y o n Dowex A G 50W-X8 (Fig. 3). Three fractions with activity were eluted. The activity of the first fraction was weaker t h a n those of the o t h e r two fractions a n d e n d o g e n o u s G A B A was eluted in the second fraction with I N pyridine as eluant. The e n d o g e n o u s inhibitor in the third fraction appeared to be basic, as it was eluted with 1 N NH4OH. These results seem to indicate that the inhibitor in the third fraction is not G A B A itself. The third fraction that c o n t a i n e d the inhibitory activity was lyophilized, dissolved in D.D.W. and further applied to a Biogel P-2 gel filtration c o l u m n (Fig. 4). As shown in this figure, the inhibitory activity was eluted in low molecular weight fractions a n d the peak
Striatum of bovine brain I 5vol D.D.W. homogenization centrifugation (100,000g, 15 min) Supernatant
I n-BuOH treatment centrifugation (8,000g, 15 rain) Aqueous phase I
boiling (100°C, 10 min) centrifugation (24,000g, 60 min) Supernatant
I lyophilization CHCI3: MeOH =2:1 treatment MeOH extract Fig. 1. Procedure for partial purification of the endogenous inhibitor of [3H]GABA binding.
Endogenous inhibitor of GABAa and GABA^ receptors
67
-0-- o-- ninhydrin staining --v......-v-.-. activity 1"120
2.0
;4(
1N HCOOH ~
r..~,~r~.w..~.,v_v,.,r.~.y..,~.~..v-w..~i.v. ~¢~-~...~.~-v'-"0 "o '¢-°~ J~
0
CII
.g e-
•
•"~
O-•-~O~-O~-O-o'•'•-o~'O
~t)
O'O'O"o'o- O
2~ number
Fraction
do
1OO
Fig. 2. Elution profile of methanol extract by anion exchange chromatography on Dowex AG l-X8. The crude methanol extract (13 g) was dissolved in 10 ml of D.D.W. and applied to a 5.0 × 15.0 cm Dowex AG l-X8 column (HCOO- form). Material was eluted first with D.D.W. at a flow rate of 2.5 ml/min and then with step wise system of 1 N HCOOH. Fractions. of 100 ml were collected. Each fraction was lyophilized and resuspended in 100 ml of D.D.W., and the effects of 100 #1 aliquots on [3H]GABA binding were examined. The position of elution of the endogenous inhibitor was determined from the percentage inhibition of total [3H]GABA binding (10 nM) (V). activity was observed just after the peak of ninhydrinpositive material.
Properties and characteristics of endogenous inhibitor After gel filtration, the active fraction (fraction 4 8 4 9 ) was subjected to thin layer chromatography
(TLC) and the chromatogram was treated with ninhydrin reagent to locate amino acids and peptides. Two ninhydrin-positive spots (Rr 0.04 and 0.17) were detected, but neither had significant inhibitory activity. However, inhibitory activity on the [3H]GABA binding was found in Rf 0.5-0.6 (Fig. 5). Rf value of .....o--- o..- activity --ua-- m- n~hydrin staining
I~
H20
Jj(
2.0 " i •
jpQ-o4,
1N NH40H
1N ~ ' t ~ 94"t~'tk/
•~ j 4 1 ~-."
.. O/" ~
\
' .~-.
' 0 o-
g
o an
o -r
g
so~
1.0
.Q
_g .o
\ ~m-L r
1'0
~-.-r~-r'%.H_j
ab
do
4b
100
Fraction number
Fig. 3. Elution profile of active fractions from Dowex AG l-X8 by cation exchange chromatography on Dowex AG 50W-X8. The inhibitor enriched fractions from Dowex AG l-X8 were lyophilized, dissolved in 10 ml of D.D.W. and applied to a Dowex 50W-X8 column (2.0 × 20 cm, H + form) equilibrated with D.D.W. Material was eluted with D.D.W., 1 N pyridine and then with 1 N NH4OH. Fractions of 20 ml were collected, lyophilized and resuspended in 20 ml of D.D.W. Then 100 pl aliquots of each fraction were assayed for activity of total [3H]GABA binding. The elution position of the endogenous inhibitor was determined as described for Fig. 2. (O).
68
ICHIMARO
•
et al.
YAMADA
O activity nitdlydrin staining
-m---a-
~OOOqDOqNDOoe~e ~--~-==---
1.0
Vo
e e e o o ~ O
eeeeee
.i GABA ~W-
o~ 0.5
i i
~/~i
5O ~.~
.-%..,'" ::::::am::::::::::::::::::: 40 50 60 70 Fraction number
Pm=ml~nl~ nl,alm =m~
~" 10
20
30
100 80
90
Fig. 4. Biogel P2 chromatography of endogenous inhibitor of Na ÷-independent [3H]GABA binding. Active fractions (fr. 28-34) from the Dowex AG 50 W-X8 column were lyophilized, dissolved in 2 ml of D.D.W. and applied to a Biogel P2 column (2.4 × 105.0cm) and the material was eluted with D.D.W. at a flow rate of 0.22 ml/min. Fractions of 3.2 ml were collected and 10 #1 portions of each fraction were subjected to binding assay. (O), percent inhibition of total [3H]GABA binding; (11), absorbance at 570 nm with ninhydrin; Vo, void volume.
authentic G A B A was 0.42, but we could not find out G A B A spot on T L C of the active fraction. Accordingly, it seems reasonable to consider that the inhibitory factor is different substance(s) from G A B A . Enzymatic digestion (trypsin and carboxypeptidase Y) and boiling (at 100°C for 10 min) of this material (fraction 48-49) had no effect on its inhibitory activity. Thus the endogenous inhibitor of G A B A receptors is apparently a heat-stable and ninhydrin negative substance.
Effect of endogenous inhibitor on [3H]GABA binding The properties of the inhibitor on G A B A receptors were studied. Baclofen-sensitive GABAB sites can be detected in Tris-HC1 containing 2.5 m M CaCI 2 and 1 0 0 # M bicuculline to suppress binding to G A B A A sites. When tested under these conditions, the endogenous inhibitor inhibited [3H]GABA binding to GABAB sites with an IC50 value of 5.0 × 10 7 g/ml (Fig. 6). On the other hand, bicuculline-sensitive GABAA sites can be detected in Tris-HC1 buffer 100
.~ 1 O0
]
< 11o
< r0- i "1-
~
5o < I-1 .7t_l i
9
li!-i. ! o
i
!
i
)
1 -~ 8
Fig. 5. TLC analysis of the active fractions (fraction 48fraction 49) from Biogel P-2 gel filtration column. Spots were detected by spraying with ninhydrin reagent. Inhibitory activities on [3H]GABA binding were expressed in column. See Materials and Methods for details.
t
8
t
7 6 5 -log (inhibitor) g/ml
4
Fig. 6. Inhibition of specific [3H]GABA binding to "A" (A) and "'B" (Q) receptor recognition sites by various amounts of endogenous inhibitor. Bindings to GABAA and GABA B sites were determined as described in Materials and Methods. Synaptosomal membranes were incubated with [3H]GABA (10 nM) for 10 min at 25°C. Amount of endogenous inhibitor was expressed by dry weight of active fraction. Points are means for three experiments performed in triplicate.
Endogenous inhibitor of GABAB and GABAA receptors
effect on the Kd value and thus its inhibitory effect may be non-competitive rather than competitive. The Bmax changed from 706 fmol per mg protein to 494 fmol per mg protein in the presence of 5.0 × 10 -7 g/ml of the endogenous inhibitor (Fig. 8).
kd 24.1 nM e~o Bmax79 fmoles
.
69
DISCUSSION
Bmax77 f m o l e ~ I
0
50 Bound (fmoles /mg
i 100 protekO
Fig. 7. Scatchard plot analysis of [3H]GABA binding to "B" receptor recognition sites in the presence (A) and absence (O) of 5 x 10-7 g/ml of endogenous inhibitor. [3H]GABA was incubated at different concentrations (10-50 nM) with synaptosomal membranes (100#g protein per tube) for 10 thin at 25°C. Non-specific binding was defined as that in the presence of 10 -3 M unlabeled baclofen. For further details, see Materials and Methods. Points are means for triplicate determinations in three experiments. (Ca 2÷ free) containing 100 # M ( - ) b a c l o f e n to suppress binding to G A B A B sites. Under these conditiotls, the endogenous inhibitor inhibited [3H]GABA binding to GABAA sites with an IC50 of 2.0 x 10 -7 g/ ml (Fig. 6). Furthermore, Scatchard plot analysis of :{3H]GABA binding to "B" receptor recognition sites demonstrated that the endogenous inhibitor in~teased the dissociation constant (Kd) without changing the maximal number of binding sites (Bmax) indicating that it caused competitive inhibition. The K~ value changed from 24.1 nM to 38.6 nM in the presence of 5.0 x 10 -7 g/ml of the endogenous inhibitor (Fig. 7). On the other hand, Scatchard plot analysis of [3H]GABA binding to " A " receptor recognition sites seemed to demonstrate that the endogenous inhibitor reduced the Bmaxwithout significant
kd 155 nM
~ . = , . . . O . = ~ m Bntax 706 f moles 0
~
3 i2 1
~
0
~
--A----~A - ~ A . kd
186nM
Bmax 494 f
moles
i
i
10o
200
Bound(f moles / mg protein)
Fig. 8, Scatchard plot analysis [3H]GABA binding to "A" receptor recognition sites in the presence (A) and absence (O) of 5.0 x 10-7 g/ml of endogenous inhibitor. For details, see Fig. 6 and Materials and Methods.
Endogenous factors that modulate receptors have been found in the central nervous system (Asano and Spector, 1979; Guidotti et al., 1983; Zaczek et al., 1983; Akagawa et al., 1984; Atlas and Burstein, 1984; Rahavi et al., 1985; Diaz-Arrastia et al., 1985). We also reported the presence of a low molecular weight modulator of dopamine receptors in bovine brain striata (Hirata et al., 1983). There are also reports of the presence of endogenous modulators or inhibitors of bicuculline sensitive GABAA receptors (Toffano et al., 1978; Yoneda and Kuriyama, 1980; Kuroda et al., 1984). In this study, we demonstrated the presence of an endogenous inhibitor(s) of GABAA and GABAa receptors in bovine brain striata. This inhibitor(s) was not G A B A itself, since it was completely separated from endogenous G A B A on both Dowex 50W-X8 cation exchange and Biogel P-2 columns. Furthermore, by TLC we could not find out presence of G A B A in our inhibitor preparation and the inhibitory activity showed different location from that of GABA. The size of this endogenous substance was estimated as less than 500 Da by gel filtration on Biogel P-2. Our results also clearly indicated that the endogenous substance was distinct from the protein type of inhibitor named GABA-modulin. Bowery and his colleagues classified G A B A receptors into GABAA and GABAB classes (Hill and Bowery, 1981; Bowery et al., 1983). We examined whether the endogenous inhibitor inhibited [3H]GABA binding to GABAA or GABAB sites. We found that it inhibited the binding of [3H]GABA not only " A " receptor recognition sites but also "B" receptor recognition sites. Scatchard plot analysis of the data revealed that its inhibitory effect on [3H]GABA binding to " A " receptor recognition sites was non-competitive, whereas its inhibitory effect on [3H]GABA binding to "B" receptor recognition sites was competitive. The inhibitory action can be distinguished from the inhibition produced by G R I F (Yoneda and Kuriyama, 1980), which decreases the affinity of [3H]muscimol for GABAA receptor sites. The chemical structure of the endogenous inhibitor is unknown, but our results showed that it is a ninhydrin-negative, basic, heatstable and low molecular weight substance. This inhibitor had no significant influence on specific
70
ICHIMARO YAMADA et al.
bindings o f [3H]mepyramine, [3H]QNB and [3H]apomorphine. Accordingly, these results suggest that the e n d o g e n o u s i n h i b i t o r functions as a specific ligand for b o t h G A B A s a n d G A B A a receptors. Electrophysiological studies are n o w in progress on the physiological role of the e n d o g e n o u s inhibitor. Acknowledgements--We thank Mrs Mieko Nakamura for help in preparation of this manuscript and Mr Masamitu Sakamori (Research Laboratories, Yoshitomi Pharmaceutical Industries Ltd) for assistance in receptor binding assay. The work was supported by a Grant-in-Aid for Scientific Research form Ministry of Education, Science and Culture of Japan.
REFERENCES
Akagawa K., Hara N. and Tsukada Y. (1984) Partial purification and properties of the inhibitors of Na,KATPase and ouabain-binding in bovine central nervous system. J. Neurochem. 42, 775-780. Asano T. and Spector S. (1979) Identification of inosine and hypoxanthine as endogenous ligands for the brain benzodiazepine binding sites. Proc. Natn. Acad. Sci. U.S.A. 76, 977-981. Atlas D. and Burstein Y. (1984) Isolation of an endogenous clonidine-displacing substance from rat brain. FEBS Lett. 170, 387-390. Bowery N. G., Hill D. R. and Hudson A. L. (1983) Characteristics of GABA B receptor binding sites on rat whole brain synaptic membrane. Br. J. Pharmac. 78, 191 206. Bowery N. G., Price G. W., Hudson A. L., Hill D. R., Wilkin G. P. and Turnbull M. J. (1984) GABA receptor multiplicity. Neuropharmacology 23, 219 231. Chang L. R. and Barnard E. A. (1982) The benzodiazepine/GABA receptor complex, molecular size in the brain synaptic membrane and solution. J. Neurochem. 39, 1507 1518. Diaz-Arrastia R., Ashizawa T. and Appel S. H. (1985) Endogenous inhibitor of ligand binding to the muscarinic acetylcholine receptor. J. Neurochem. 44, 622628. Gavish M. and Snyder S. H. (1981) 7-Aminobutyric acid and benzodiazepine receptors, co-purification and characterization. Proc. Natn. Acad. Sci. U.S.A. 78, 1939- 1942.
Guidotti A., Forchetti C. M., Corda M. G., Konkel D., Bennet C. D. and Costa E. (1983) Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc. Natn. Acad. Sci. U.S.A. 80, 3531-3535. Hill P. R. and Bowery N. G. (1981) [3H]Baclofen and [3H]GABA bind to bicuculline insensitive GABA a sites in rat brain. Nature, Lond. 290, 149 152. Hirata A., Fujita N., Saito K. and Yoshida H. (1983) Endogenous regulator of dopamine receptors. Jap. J. Pharmac. Supplementum 33, 219 p. Neuroehem. Int. In Press. Kuroda H., Ogawa N., Nukina I. and Ota Z. (1984) An endogenous inhibitor of GABA receptor binding. Neurochem. Res. 9, 21 -27. Leeb-Lungberg F. and Olsen R. W. (1983) Heterogeneity of benzoidiazepine receptor interaction with 7-aminobutyric acid and barbiturate receptor sites. Molec. Pharmac. 23, 315 325. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. McGeer P. L. and McGeer E. G. (1981) Amino acid neurotransmitters. In Basic Neurochemistry, 3rd edn (Albers, R. W., Siegel G. J., Kotzman R. and Agranoff B. W.. eds), pp. 233 253. Little, Brown & Co., Boston. Rahavi M., Ventra 1. and Sarne Y. (1985) Demonstration of endogenous "imipramine-like" material in rat brain. L(fe Sci. 36, 687~593. Siegel E. and Barnard E. A. (1984) A ),-aminobutyric acid/ benzodiazepine receptor complex from bovine cerebral cortex. J. biol. Chem. 259, 7219-7223. Toffano G., Guidotti A. and Costa E. (1978) Purification of an endogenous protein inhibitor of the high affinity binding of l'-aminobutyric acid to synaptic membranes of rat brain. Proe. Natn. Acad. Sci. U.S.A. 75, 4024~4028. Wojcik W. K. and Neff N. H. (1984)),-Aminobutyric acid B receptors are negatively coupled to adenylate cyclase in brain, and in the cerebellum these receptors may be associated with granule cells. Molec. Pharmac. 25, 24-28. Yoneda Y. and Kuriyama K. (1980) Presence of low molecular weight endogenous inhibitor of 3H-muscimol binding in synaptic membranes. Nature, Lond. 285, 670 673. Zaczek R., Koller K., Cotter R., Heller D. and Coyle J. T. (1983) N-Acetylaspartylglutamate: an endogenous peptide with high affinity for a brain "glutamate" receptor. Proc. Nam. Acad. Sci. U.S.A. 80, 1116-1119.