Br. J. Pharmac. (1983), 80, 355-364

Characterization of the binding of DL-[3H]-2-amino-4-phosphonobutyrate to L-glutamate-sensitive sites onrat brain synaptic membranesS.P. Butcher, J.F. Collins* & P.J. Roberts

Department of Physiology and Pharmacology, Medical and Biological Sciences Building, University ofSouthampton, Southampton, S09 3TU and Department of Chemistry*, City of London Polytechnic, 31 JewryStreet, London EC3N 2EY

1 The binding of DL-[3,4-3H] 2-amino-4-phosphonobutyric acid DL[3H]-APB to rat whole brainsynaptic membranes was investigated.2 Binding was linear with membrane protein concentration, and optimal at physiological pH andtemperature. The association rate was rapid, achieving equilibrium within 10 min. Prolongedincubations (>20 min) revealed additional sites, which apparently possessed identical bindingcharacteristics to those detected with 10 min incubations.3 Binding of DL-[3H]-APB was enriched in synaptic membrane fractions, and assessment of theregional distribution, indicated greatest binding in those areas with a rich glutamatergic innervation.4 The binding of DL-[3H]-APB inHEPES-KOH buffer exhibited an absolute requirement for Cl-.The addition of Ca2l resulted in a further enhancement of binding.5 Saturation analysis revealed the presence of specific glutamate-sensitive DL-[3H]-APB bindingsites, with a KD = 1.26 p4M and B,,. = 12.08 pmol mg-1 protein. A Hill plot revealed a slope slightlygreater than unity, which could possibly be a reflection of a contribution to binding of a further sitewhich is relatively insensitive to glutamate. Analysis of 60 min incubation data indicated an

approximately 3 fold increase in the capacity of the system, but a relatively unchanged KD.6 Examination of the pharmacological specificity of binding, showed that for both agonist andantagonist molecules, the L-enantiomers were invariably more active than the D-forms. Forexample, the L-(+)-2-amino-4-phosphonobutyrate isomer was 15 times more active than theD-(-)-form in inhibiting the binding of DL-[3H]-APB. This is in close agreement with the ability ofthese compounds to produce depression of synaptic transmission. The most potent inhibitor ofbinding was quisqualate. It is suggested that APB may interact with a quisqualate-preferring class ofexcitatory amino acid receptors, possibly localised predominantly on presynaptic terminals.

Introduction

Systematic neuropharmacological studies have indi-cated the presence of at least three types of receptorwith which the neurotransmitters glutamate, aspar-tate and other endogenous dicarboxylic amino acidsmay interact. These receptors are activated preferen-tially by the analogues N-methyl-D-aspartate(NMDA), kainate and quisqualate (Watkins,198 la). While the NMDA site has been well charac-terized pharmacologically, there is a lack of suitablechemical probes for investigating the kainate andquisqualate receptors. Indeed, it is still not clear howmany receptor types, or subclasses fall within thenon-NMDA category. L-Glutamate diethylester(GDEE) has been reported to antagonize quisqual-

ate and glutamate, but not kainate responses in catspinal neurones (Davies & Watkins, 1979; McLen-nan & Lodge, 1979). However, this compound is oflow potency and is often considered to be a ratherunreliable antagonist of uncertain mode of action.The compound y-D-glutamyl-glycine is able to dis-criminate between quisqualate and kainate receptorsin the cat spinal cord, depressing spinal responseselicited by the latter compound (Davies & Watkins,1981). The most potent antagonist at quisqualatereceptors is cis-2,3-piperidinedicarboxylate, al-though this substance also blocks NMDA and kain-ate receptors.

Highly potent and specific antagonists for the

c) The Macmillan Press Ltd 1983

356 S.P. BUTCHER etal.

NMDA receptor have been revealed amongst aseries of w-phosphono-a-carboxylic amino acids,notably, 2-amino-5-phosphonopentanoate and thecorresponding heptanoate analogue (Evans & Wat-kins, 1981; Watkins, 1981a; Evans, Francis, Jones,Smith & Watkins, 1982). A lower homologue, DL-2-amino-4-phosphonobutyrate (APB) (in which theu-carboxylate group of glutamate is replaced byphosphonate) was originally reported to possessweak glutamate-like agonist activity (Curtis & Wat-kins, 1965; Watkins, Curtis & Brand, 1977), while inseveral invertebrate species, APB depressedglutamate-induced depolarizations (Cull-Candy,Donnellan, James & Lunt, 1976; Dudel, 1977).More recent studies have demonstrated antagonismby APB at vertebrate excitatory amino acid receptors(Davies & Watkins, 1979; Hori, Auker, Braitman &Carpenter, 1981). However, this appeared to berather non-selective, since APB was effective inblocking responses elicited by glutamate, aspartate,kainate, quisqualate and NMDA. This effect at post-synaptic amino acid receptors seems to be a propertyof the D(-)-isomer (Davies & Watkins, 1982; Evanset al., 1982). In contrast, L(+)-APB has a potent andstereoselective synaptic depressant action (Davies &Watkins, 1982; Evans et al., 1982; Koerner & Cot-man, 1981) which does not appear to be associatedwith blockade of postsynaptic amino acid receptors.At somewhat higher concentrations than thosenecessary for synaptic depressant effects, the L-isomer elicits 2-amino-5-phosphonopentanoate-sensitive depolarization, indicating some NMDA re-ceptor agonist activity (Evans et al., 1982).With the recent availability of highly-labelled DL-

APB (Monaghan, Fagg, Mena, Nieto-Sampedro,McMills, Chamberlin & Cotman, 1982), we haveinvestigated the characteristics of the binding sites forthis substance on rat brain synaptic membranes, in anattempt to gain additional information concerningnon-NMDA type excitatory amino acid receptors. Apreliminary account of this work has appeared as ashort note (Butcher, Roberts & Collins, 1983).

Methods

Synaptic membranes

Albino Wistar rats (250-300g, either sex) werekilled by decapitation and the brains removed rapid-ly. Synaptic membranes were prepared by a modifi-cation of our procedure (Sharif & Roberts, 1980).Briefly, brains were homogenised in a Teflon-glasshomogeniser (0.1 mm clearance, 5 strokes, approx.2,000 r.p.m.), in 20 vol 0.32 M sucrose, buffered with5 mM HEPES-KOH (pH 7.4). The homogenate wascentrifuged for 10 min at 1,000 gin a Beckman J2-21

centrifuge, and the pellet discarded. The supernatantwas centrifuged again at 17,000 g for 20 min, and theresulting P2 pellet was resuspended in 5 mM HEPES-KOH buffer (pH 7.4) with a tight-fitting glass-glasshomogeniser, and the suspension placed on ice for10 min. This procedure was followed by centrifuga-tion at 17,000 g for 20 min, resuspension of the pel-let; in fresh buffer and further centrifugation. Thefluffy 'buffy coat' was carefully removed from thesurface of the pellet; resuspended in 5 mM HEPESbuffer and centrifuged at 50,000 g for 20 min. Thiswashing procedure was repeated a further twice, andthe synaptic membrane-enriched pellet finally resus-pended in 50 mM HEPES-KOH buffer (pH 7.4) con-taining 2.5 mM CaCl2.

Subcellularfractionation

In some experiments, whole rat brains werehomogenised with a Teflon-glass homogeniser in20 vol 0.32 M sucrose, buffered with 5 mM HEPES(pH 7.4), and a P2 pellet prepared as describedabove. This was resuspended in a small volume (lessthan 1 ml) of sucrose, and layered onto a biphasicgradient consisting of 7 ml 0.8 M sucrose, and7 ml 1.2 M sucrose, buffered with 5 mM HEPES-KOH (pH 7.4). After centrifugation for 60 min at100,000 g in a Beckman SW 27 swing-out rotor,crude myelin, synaptosomal, and mitochondrial frac-tions were harvested, and lysed in 5 mM HEPES-KOH buffer, using a glass-glass homogeniser. Afterstanding on ice for 10 min, membranes were pelletedby centrifugation at 50,000 g for 20 min. Pellets wereresuspended in buffer, and recentrifuged 3 timesprior to final resuspension in buffer, and assay forbinding of APB.

DL-pH]-2-amino-4-phosphonobutyrate bindingassay

Assays were carried out in 1.9 ml polypropylenemicrofuge tubes (Elkay Inc., MA, U.S.A.). To eachtube was added 25 fl of DL-[3,4-3H]-2-amino-4-phosphonobutyric acid (26.6 Ci/mmol) plus, in in-hibitor studies, 25 jl of a solution of the substanceunder test, or buffer control. Non-specific bindingwas defined by total radioactivity bound minusradioactivity bound in the presence of 1 mM L-glutamate in the medium. In experiments where asingle, fixed DL-[3H]-APB concentration was re-quired, this was usually 30 nM (10 nM label, dilutedwith 20 nM unlabelled DL-APB). The binding assaywas initiated by addition of 0.5 ml of membranesuspension (in 50mM HEPES-KOH, pH 7.4, with2.5 mM CaCl2) and continued, usually for 10 min at37°C in a shaking water bath. Tubes were then cen-trifuged for 30 s in a Beckman Microfuge B, and the

DL-2-AMINO-4-PHOSPHONOBUTYRATE BINDING 357

supernatant aspirated immediately. Pellets wererinsed carefully with 1 ml buffer, the tube tips cut off,and the membranes solubilised overnight in scintilla-tion vials containing 2% SDS. Bound radioactivitywas determined by liquid scintillation counting fol-lowing the addition of 8 ml scintillant (xylene,5.34 ml; synperonic NXP, 2.66 ml; PPO, 32 mg anddimethyl POPOP, 4 mg) in a Beckman LS 7500 li-quid scintillation spectrometer with external standar-disation and automatic quench correction.

Compounds and chemicals

DL-[3H]-APB was supplied by New England Nuc-lear, Boston, MA, U.S.A. Scintillation fluors werefrom Koch-Light; synperonic NXP from ICI plc andstandard reagents from BDH Ltd., Poole, Dor-set. N-methyl-D-aspartate, y-D-glutamylglycine, D-a-aminoadipate, L-a-aminoadipate, DL-a-amino-suberate and DL-a-aminopimelate were gifts from DrJ.C. Watkins (Bristol). a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) was fromDr P. Krosgaard-Larsen (Copenhagen); (±)-ibotenate from Prof C.H. Eugster (Zurich) and 1-hydroxy-3-aminopyrrolid-2-one (HA-966) from DrH.J. Broxterman (Leiden). All other amino acidanalogues were synthesised by ourselves, or purch-ased from Sigma U.K., or Cambridge ResearchBiochemicals. The isomers of APB had the following[a]546 values (determined in water): D-(-)-APB,-11.8° and L-(+)-APB, + 10.80.

Results

Table 1 Effects of pH and temperature onspecific DL-[3H]-2-amino-4-phosphonobutyrate(DL-[3H]-APB) binding to whole brain synapticmembranes

Treatment

pH 6.07.07.48.09.0

Temperature (°C) 0223750

Specific binding(fmolmg 1 protein)

105 ± 17178 ± 36251± 27154±1168± 19

39±61256±79299± 28

3 ±28

Freshly-prepared, whole rat brain synaptic mem-branes were incubated for 10min with DL-[3H]-APB (30 nM) in the absence, or presence of L-glutamate (1 mM) as described in the text. Resultsare means ± s.e.mean from duplicated experimentsperformed in quadruplicate.

almost certainly a consequence of binding site denat-uration. The non-specific binding component wasessentially constant under each of these conditions. Itis worth noting, that, in common with L-glutamatebinding (Foster & Roberts, 1978), specific binding ofDL-[3H]-APB was substantially reduced followingfreezing of the synaptic membranes.

Dependence ofbinding on membraneproteinconcentration

The specific binding of DL-[3H]-APB (30 nM), whichrepresented approximately 30% of the total binding,was found to be linear with membrane protein con-centration over a wide range (16-780 pg protein). Atthe highest protein concentration, this representedapproximately 230 fmol DL-[3H]-APB specificallybound. In all routine experiments, protein concentra-tions were maintained in the range 200-600 fig perassay.

Effects ofvaryingpHand temperature

Specific binding of DL-[3H]-APB was evident overthe pH range 6.0-9.0. However, it exhibited a dis-tinct optimum between pH 7.0 and 8.0 (Table 1).The incubation of membranes with 30 nM DL-[3H]-

APB for 10 min at various temperatures, indicatedgreatly reduced binding at 0°C and 50°C, comparedwith 22°C and 37°C. The decreased binding at 50°C is

Time course ofbinding: association and dissociation

The time dependency of specific DL-[3H]-APB bind-ing was biphasic. Initially, binding was extremelyrapid, attaining an equilibrium value of approximate-ly 240 fmol bound mg-1 protein, within 10 min. Thisequilibrium value was maintained for at least a furth-er 10 min, after which, binding increased again sharp-ly, reaching a new equilibrium value of approximate-ly 600 fmol mg-I protein after 50-60 min incubation(Figure 1). With these longer incubations, someclumping and precipitation of membrane particleswas evident. This was never observed during the timeperiod over which the initial equilibrium was estab-lished.The dissociation of bound DL-[3H]-APB was rapid

and followed a typical exponential decay curve, witha half-life of approximately 90s (Figure 2). After20 min however, there was still significant detectableresidual binding, which may reflect the changes oc-curring in the synaptic membranes during long incu-bations.

358 S.P. BUTCHER etal.

Table 2 Effects of ions on specific DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB)binding binding

Ions added(2.5 mM)

0 10 20 30 40 50 60Time (min)

Flgure 1 Time course of association of DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB) to wholerat brain synaptic membranes. Synaptic membranes(200-600 lAg protein) were incubated for various timesat 37C in HEPES-KOH buffer (pH7.4) containing2.5 mM CaCl2 and 30 nM DL-[3H]-APB as described inthe text. Specific APB binding was defined as thatdisplaced by 1 mM L-glutamate. Results are means withs.e.mean of quadruplicate determinations from 4 inde-pendent experiments. Note the biphasic nature of thecurve.

Effects ofCa2", Cl and other ions onDL-pH]-APBbinding

Incubation of synaptic membranes for 10 min withDL-[3H]-APB, in the absence of any added ions otherthan K+ (50mM HEPES-KOH buffer, pH 7.4), re-

sulted in minimal or non-detectable levels of specificbinding (Table 2). In the presence of both Ca2l and

250

CLnc 200_

150

'_ E l00

50

4)

0 5 10 15 20Time (min)

FIgure 2 Dissociation of DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB) from specific bind-ing sites on synaptic membranes. Synaptic membraneswere incubated at 37°C with 30nM DL-[3H]-APB as

described in the text. After a 10 min incubation, excessunlabelled (1 mM) L-glutamate was added and, at vari-ous times, specific binding was determined. Results aremeans with s.e.mean of quadruplicate determinationsfrom 2 independent experiments.

Specific binding(fmol mg-1 protein)

NoneCalcium chlorideCalcium acetateAmmonium chlorideAmmonium acetateMagnesium chlorideManganese chloride

Whole rat brain synaptic membranes were incu-bated in HEPES-KOH buffer (pH 7.4) for 10 minwith DL-[3H]-APB (30 nM), in the absence orpresence of added ions. Specific binding was deter-mined as described in the text. Results aremeans ± s.e.mean from at least 3 experiments, per-formed in quadruplicate.

Cl- (as CaCl2, 2.5 mM), specific binding of 30nMDL-[3H]-APB was approximately 250 fmol mg-'1protein. In view of the report that Cl- stimulatesL-glutamate binding to synaptic plasma membranes,and that Ca2" acts only in the presence of Cl- toenhance this response further (Mena, Fagg & Cot-man, 1981), we investigated the binding of DL[3H]-APB to synaptic membranes in the presence of anumber of ionic species (Table 2). Indeed, Cl- alone(NH4Cl compared with CH3COONH4, both at2.5 mM) was able to enhance binding, and thus ap-pears to promote the further enhancement seen inthe presence of Ca2". Calcium acetate had a minimalstimulatory action. Magnesium, and especially man-ganese were able partially to mimic the effects ofcalcium (all as the chloride salt).

Regional specificity ofbinding

Synaptic membranes from a number of regions of therat central nervous system were prepared as de-scribed for whole brain, and the specific binding ofDL-[3H]-APB determined (Table 3). Compared withbinding to whole brain membranes, specific bindingwas enriched particularly in striatum and hippocam-pus, and was least in the pons/medulla, where it wasapproximately one fifth of that occurring in theformer two regions.

Subcellulardistribution ofDL-pH]-APB binding sites

Subcellular fractionation of whole rat brain homoge-nates, resulted in an initial enrichment in the P2fraction, which was further increased in the synapticmembrane fraction. Binding in the P1, myelin and

o, 600C

* c 500

0- 4.400

I-

005o E200

a 100,) a

16± 19251±1833± 6102±1818± 33

127± 32185 ± 25

DL-2-AMINO-4-PHOSPHONOBUTYRATE BINDING 359

Table 3 Regional specificity of - DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB)binding

Specific binding(fmol mg- 1 protein)Neural-tissue

StriatumHippocampusCortexCerebellumPons/medullaSpinal cordWhole brain

559± 78574± 90467± 83203 ±79129 ± 1744 ± 67279± 30

Following killing of the animals, the above regionswere rapidly dissected out and homogenized in20 vol buffered 0.32M sucrose. Synaptic mem-branes were prepared simultaneously for each ofthe regions as described in the text, and the specificbinding of 30 nM DL-[3H]-APB determined duringa 10 min incubation. Results are means ± s.e.meanfrom 2 experiments performed in quadruplicate.

mitochondrial fractions was variable between prep-arations, but was essentially minimal (Table 4).

Saturability ofDL-pH]-APB binding

In view of the earlier observation that the time courseof DL-[3H]-APB binding exhibited biphasic kineticswith an initial rapid establishment of equilibrium,followed by a further increase in binding, studieswere carried out with incubations of both 10 and60 min duration. Analysis of untransformed specificbinding data obtained over the concentration range

Table 4 Subcellular distribution of specific DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB)

Specific binding(fmol mg-1 protein)Tissue fraction

P1 pelletP1 supernatantP2 supernatantP2 pelletCrude myelinMitochondrialSynaptic membraneWashed synaptic membranes

67±2223±11118±2964± 823 ± 17257±24283± 96

Whole rat brains were fractionated as described inthe text, and the binding of DL-[3H]-APB(30 nM) determined. Results are means +s.e.mean of 2 experiments (performed in quadru-plicate) from 5 or 6 pooled brains.

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[DL-[ H]-APB] (liM)Figure 3 (a) Saturation of DL-(3H)-2-amino-4-phosphonobutyrate (DL-[3H]-APB) binding to wholerat brain synaptic membranes. Synaptic membraneswere incubated at 37°C for 10 min with a range ofconcentrations of DL-[3H]-APB. Specific APB bindingwas defined using 1 mM L-glutamate. Results are meansof quadruplicate determinations from 2 independentexperiments. KD and B,,. were calculated using acomputer-derived curve fitting process, based on thatdescribed by Wilkinson (1961). The inset shows a Hillplot of the binding data. (b) Saturation of DL-[3H]-APBbinding to whole rat brain synaptic membranes incu-bated at 37° for 60 min. Otherwise, conditions wereidentical to those described for (a).

10nM-20fLM DL-[3H]-APB revealed the apparentpresence of a single, saturable population of bindingsites (Figure 3a and b). For the 10 min incubation,sites were detected with a KD = 1.26 ± 0.07 luM andB,X = 12.08 ± 0.5 1 pmol mg-1 protein. After 60 minincubation, the values found were KD = 1.09±0.06 t4M and B,,. = 39.35 ± 0.945 pmol mg-1 protein(means± s.e.mean of quadruplicate determinationsperformed in duplicate). Thus, the increased bindingobserved following prolonged incubation of the

360 S.P. BUTCHER etal.

a

140 - (±) -APB - D-Homocysteate Co (±)-AP5 o (±)-AP7C * (-)-APB 0 L-Cysteinesulfinate * (-)-AP5 -)-AP7._ 0 (+)-APB * 0 L-Cysteate O(+)-AP5 0 (+)-AP7

<< 120 * QisQala 0 0 L-HomocysteateS120 - o AMPA 0 - O L-Asp _0_

2~80 0

C.260

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0.

O0 -

0

-7 -6 -5 -4 -3 -7 -6 -5 -4 -3 -7 -6 -5 -4 -3 -7 -6 -5 -4 -3

log [Inhibitor] (M)

b

re140 * Quisaualatei (+)-Ibotenate o L-Y -Aminoadipate * Fluoroglutamate0 D-Glutamate 0 0 (±)-AP6 0Dn-t -Aminoadipate 0 DL-f-AminopimelateA L-Glutamate 2 D-Asp2 DL-nY-Aminoadipate UDL-an-Aminosuberarat

h n120 o AMPAa L-Asp

A5100 0z

80 0

C.2060-

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log [Inhibitor] (m)

Flgure 4 (a and b) The inhibition of DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB) binding to rat brainsynaptic membranes by excitatory amino acid analogues. Synaptic membranes were incubated for 10 min at 370Cwith 30 niM DL-[3H]-APB in the presence of a wide range of inhibitory compounds as described in the text. Thecompound under test was added simultaneously with the labelled ligand. Results are means of quadruplicatedeterminations from at least two independent experiments. IC50 values (that concentration of inhibitor that reducedbinding by 50%) were read directly from the plots.AP5: 2-amino-5-phosphonopentanoate; AP6: 2-amino-6-phosphonohexanoate; AP7: 2-amino-7-phosphono-heptanoate; AMPA: aE-amino-3-hydroxy-5-methylisoxazole-4-propionate.

DL-2-AMINO-4-PHOSPHONOBUTYRATE BINDING 361

synaptic membranes, appears to be wholly due to anincrease in the number of binding sites exposed to theligand, with no change in their affinity. Hill plots forboth sets of data (Figure 3a and b insets) revealedslopes of 1.26±0.01 and 1.35 ±0.03 for the 10 and60 min incubations respectively. Although these val-ues were greater than unity, they were insufficientlydifferent to be confident that there is siteheterogeneity, or interactions between binding sites.Of interest was the finding that a 10 min incubation ofDL-[3H]-APB in the presence of Cl- but not Ca2+(binding assay medium of HEPES-KOH + 2.5 mMNH4Cl) gave a KD of approximately 1.44 ELM, and aB,, = 7.60 pmol mg 1 protein (data not shown), in-dicating that the effect of the further addition of Ca2'is to increase the number of sites available for in-teraction with the ligand.

Pharmacological specificity ofDL-pHI-APB binding

The pharmacological specificity of the binding ofDL-[3H]-APB was investigated by incubating synap-tic membranes with ligand (30 nM) for 10 min at 37°Cin the presence of a range of concentrations(0.3 AM- 1.0 mM) of the inhibitory compound undertest. Log concentration-% inhibition curves wereconstructed (Figure 4a and b) and IC50 values deter-mined (Table 5).The L-isomer of APB was approximately 15 times

more potent than the D-form in inhibiting binding.For a number of agonists such as homocysteate,glutamate, and aspartate, L/D-isomer activity ratiosof around 6, 11 and 9 respectively, were obtained.The binding site was not sensitive to kainate or to anumber of specific ligands for other receptors, viz.carbachol, glycine, y-aminobutyric acid (GABA) andnoradrenaline. N-methyl-D-aspartate (or N-methyl-DL-aspartate) and quinolinic acid, were very weaklyactive in inhibiting DL-[3H]-APB binding.With specific excitatory amino acid receptor an-

tagonists, the pattern of binding was confusing. L-(+)-APB was highly active, and in common with theL-isomers of sulphur-containing amino acids, andwith quisqualate, inhibited [3H]-APB binding to asubstantially greater extent than the 100% definedby 1 mM L-glutamate (Figure 4a and b). The isomersof other phosphono amino acid derivatives, such as2-amino-5-phosphonopentanoate, and 2-amino-7-phosphonoheptanoate, all exhibited some activity.Again, it is striking that the L-(+)-isomers were themore active. A similar pattern emerged for a-aminoadipate and a-aminosuberate. A number ofother antagonists were also investigated, and werefound to possess either little (L-glutamate diethyles-ter) or no (cis-2,3 piperidine dicarboxylate and -y-D-glutamylglycine) activity.

Table 5 Inhibition of DL-[3H]-2-amino-4-phosphonobutyrate (DL-[3H]-APB) binding to ratbrain synaptic membranes by excitatory aminoacid receptor agonists and antagonists

Agonist

QuisqualateL-HomocysteateL-GlutamateL-CysteateD-HomocysteateL-AspartateL-Cysteine sulphinateDL-Fluoroglutamate(±)-IbotenateD-Glutamatea-Amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)D-Aspartate

AntagonistDL-a-AminosuberateL(+)-APBL-xa-AminoadipateDL(±)-APBDL- a-AminoadipateDL(±)-2-Amino-6-phosphonohexanoateD-a-AminoadipateDL-a-AminopimelateL(+ )2-Amino-7-phosphonoheptanoateL( +)-2-Amino-5-phosphonopentanoateD(-)-APBDL(±)-2-Amino-7-phosphonoheptanoateDL(±)-2-Amino-5-phosphonopentanoateD(-)-2-Amino-7-phosphonoheptanoateD(-)-2-Amino-5-phosphonopentanoate

IC50(rIM)0.3981.211.704.006.306.977.087.9010.0018.10

25.2456.20

1.582.243.003.616.13

14.4016.1816.5118.9620.0030.0632.6039.80

100126

Weakly active (IC5o> 100 um)N-methyl-D-aspartate, quinolinic acid, (±)-2-amo-3-phosphonopropionate, cis-2,3-piper-idinedicarboxcylate, DL-a, 8-diaminopropionate,7-D-glutamyl glycine.

Inactive (IC5o> 1 mM)Kainate, dihydrokainate, 1-hydroxy-3-amino-pyrrolid-2-one, carbachol, glycine, GABA,noradrenaline, phenobarbitone.Syna3ptic membranes were incubated with 30nMDL-[ HI-APB in the absence or presence of a widerange of concentrations (0.3 riM- 1.0 mM) of thecompound under test, as described in the legend toFigure 4. IC50s (the concentration required to pro-duce 50% inhibition for that compound) were readdirectly from the log concentration/percentage in-hibition curves.

Discussion

The mechanism of action of DL-APB has been thesubject of debate since the compound was first shown

362 S.P. BUTCHER etal.

to be an antagonist at the locust neuromuscular junc-tion (Cull-Candy et al., 1976). Early experimentswith vertebrate preparations indicated that on cere-

bral and cerebellar neurones in the rat, DL-APB was

a glutamate-like agonist (Bioulac, De Tinguy-Moreaud, Vincent & Neuzil, 1979), whileionophoretic application of DL-APB to cat spinalneurones failed to reduce excitation by glutamate(Watkins et al., 1977). Recent resolution of the D-(-)- and L-(+)-isomers of APB has helped to clarifythe situation, and it is now generally accepted that theD-(-)-isomer is indeed a rather weak and probablynon-selective antagonist at postsynaptic excitatoryamino acid receptors (Davies & Watkins, 1982; Horietal., 1981). The actions of the L-(+)-isomer remainenigmatic: it has been reported by several groups thatthe compound possesses a potent and stereoselectivesynaptic depressant action (Koerner & Cotman,1981; Davies & Watkins, 1982). It has been prop-

osed that L-(+)-APB may act by specifically inhibit-ing the release of an excitatory amino acid transmit-ter, acting at non-NMDA receptors, or alternatively,by antagonizing the postsynaptic effects of an as yetunidentified transmitter (Davies & Watkins, 1982).Collingridge, Kehl & McLennan (1983a) have foundthat on rat hippocampal CA1 neurones, the effect ofL-APB was typically to enhance amino acid-inducedresponses, and occasionally to excite cells by itself. Inaccordance with the excitatory effects reported in thefrog and rat spinal cords (Evans et al., 1981), theagonist effects were sensitive to both D-APB and2-amino-5-phosphonopentanoate, implicating an in-volvement of NMDA receptors. Similar results werefound at the Schaffer collateral-commissural path-way, with both isomers of APB being weakly active indepressing the e.p.s.p. (Collingridge, Kehl & McLen-nan, 1983b). Neurochemical studies have shown thatDL-APB is without effect on high-affinity glutamateuptake (Balcar & Johnston, 1972; Roberts & Wat-kins, 1975) (but see Vincent & McGeer, 1980).Thus, this is unlikely to be the mechanism by whichresponses to excitatory amino acids are potentiatedby APB.

In terms of neurochemically- (as distinct fromelectrophysiologically) defined receptor sites, con-

siderable information has accumulated. Foster &Roberts (1978) reported that DL-APB inhibited theNa+-independent binding of L-[3H]-glutamate to ratcerebellar synaptic membranes, in a Cl--containingmedium. In a later study, where a postsynaptic re-

sponse was measured (the ability of excitatory aminoacids to increase tissue levels of cyclic GMP), DL-APB was an effective, though weak antagonist ofglutamate and aspartate, and had rather less effect onthe responses elicited by NMDA and kainate(Roberts, Foster, Sharif & Collins, 1982). With re-

gard to glutamate binding studies, an important ob-

servation has been that chloride and calcium ionsapparently separate distinct receptor populations(Fagg, Foster, Mena & Cotman, 1982; 1983; Mena etal., 1981). The Cl-/Ca2+-dependent glutamate bind-ing was inhibited potently by L-APB, which was some15 times more active than the D-isomer in this re-spect. This latter binding site does not appear toconform to the current model for excitatory aminoacid receptors (Watkins, 1981a). It is insensitive toboth NMDA and kainate, while quisqualate and (theproposedNMDA receptor-preferring agonist) ibote-nate, exhibited somewhat surprisingly similar poten-cies, and yielded non-linear Scatchard plots in thepresence of CaCl2. Maximally, 70-75% of glutamatebinding was inhibited with Kis of 0.1 -0.5 !LM, and theremaining 25% with much lower affinity(40-80 mM).Because L-glutamate is a 'mixed agonist' (Watkins

& Evans, 1981), interacting with several subpopula-tions of receptors in binding experiments, attempts toinvestigate the sites of action of APB are most likelyto be resolved using labelled APB itself, and prefera-bly, the individual isomers. In this study, we haveshown that DL-[3H]-APB binds specifically to L-glutamate-sensitive sites on rat whole brain synapticmembranes, with a KD = 1.3 !LM. This value corres-ponds well with the Ki of 16 jtM for APB on gluta-mate binding (Fagg et al., 1982). DL-[3H]-APB bind-ing was optimal at physiological pH and temperatureand was totally dependent on Cl- and Ca2+, and inthe absence of these ions, only minimal binding (pos-sibly attributable to residual ions) was detectable.Binding assays performed in full physiologicalmedium (Krebs-Ringer, buffered with HEPES,pH 7.4) revealed no binding in excess of that ob-served with added Cl-/Ca2+.The distribution of binding, both regional and

subcellular, was wholly consistent with its being as-sociated with membranes of synaptic origin derivedfrom neurones involved in excitatory amino acidtransmission. For example, the high level of bindingobserved in the striatum and hippocampus is compat-ible with the rich glutamatergic innervation of theseareas of the brain.A most striking observation, was the sharp

biphasic nature of the time course of binding, where-by a second binding component appeared, resultingin a second binding equilibrium with approximately30 min, or longer incubations. This was due entirelyto an increase in binding site capacity. Recent workindicates that the pharmacological characteristics ofthese sites are identical. This 'unveiling' of APBbinding sites is mediated by a calcium-dependentprocess, and may involve activation of a calcium-dependent cysteine proteinase, since p-chloromercuribenzoyl-sulphonic acid and leupeptinwere effective in preventing the second phase of

DL-2-AMINO-4-PHOSPHONOBUTYRATE BINDING 363

binding (Butcher, Roberts & Collins, unpublished).Interestingly, a similar observation has been madefor the regulation of hippocampal glutamate recep-tors (Baudry & Lynch, 1980; Vargas, Greenbaum &Costa, 1980; Baudry, Bundman, Smith & Lynch,1981) and has been proposed as a possible mechan-ism involved in long-term synaptic potentiation.The pharmacology of DL-[3H]-APB binding gen-

erally corresponds closely with that for APB-sensitive glutamate binding (Fagg et al., 1983). Inparticular, L-APB was 15 times the activity of D-APBin both studies, and excitants such as kainate andNMDA were essentially inactive. Quisqualate wasthe most active substance investigated and, in con-trast to the previous study (Fagg et al., 1983), wasmuch more potent than ibotenate. Since L-APB,quisqualate, and the sulphur-containing excitatoryamino acids were able to inhibit binding to a greaterdegree than L-glutamate, it follows that the DL-[3H]-APB binding sites are not homogeneous (notwith-standing the observed Hill slopes of close to unity forthese compounds). The L-(+)-isomers of agonistswere also more active than the D-(-)-forms in in-hibiting DL-[3H]-APB binding, and so also were theL(+)-isomers of a number of NMDA-receptor an-tagonists (where the D-(-)-isomer carries most, ifnot all of the pharmacological activity), such as 2-amino-5-phosphonopentanoate and 2-amino-7-phosphonoheptanoate.Thus, the binding site for DL-APB recognizes

primarily L-isomers, and one might be temptedto propose that it is a quisqualate class of gluta-mate receptor. A cautionary note here though,is that x-amino-3-hydroxy-5-meythylisoxazole-4-propionate (AMPA), a proposed agonist atquisqualate-preferring receptors, is a much weakerinhibitor of binding than expected. When AMPAbinding itself was determined (Honor6, Krogsgaard-Larsen, Hansen & Lauridsen, 1981), the two agonistswere equipotent. Additionally, the quisqualate-typeantagonist, L-glutamate diethylester was of low po-tency, although it is pertinent that the usefulness ofthis compound is uncertain.

Finally, it must be stated that this study has notprovided any direct information as to the localisationof DL-[3H]-APB binding sites, or concerning themechanism of the ionic specificity of binding. For thelatter, Mena et al., (1982) proposed that these bind-ing sites may be linked to a membrane Cl- ionchannel, or alternatively, that the Cl-/Ca2+-depen-dent site might represent a desensitized form of thereceptor. In view of the general observation that theL-conformations of both agonist and antagonistmolecules are significantly more potent in inhibitingDL-[3H]-APB binding than the D-isomers, we con-sider it likely that the site is agonist-preferring, andpossibly located presynaptically. In hippocampalslices, DL-APB behaves as an antagonist at glutamateautoreceptors, by preventing the auto-inhibition byglutamate of its own release (McBean & Roberts,1981). However, since in that study only the race-mate was examined, a composite effect was probablybeing observed. In the retina, L-APB was found toinhibit both acetylcholine release and the ERG b-wave some 15 times more effectively than the D-isomer (Neal, Cunningham, James, Joseph & Collins,1981). This potency ratio is in accord with our bind-ing data. However, here at least, both D- and L-APBappear to be acting primarily as agonists at a post-synaptic excitatory receptor. Current experiments inour laboratory are aimed at resolving the D- andL-[3H]-APB binding components, and the elucida-tion of the cellular localisation of these sites.

This work was supported by a research project grant toP.J.R. from the Science and Engineering Research Council.We thank Dr Dick Young (New England Nuclear) for hisinterest and help, and Dr J.C. Watkins (Bristol), Dr P.Krogsgaard-Larsen (Copenhagen) and Professor C.H.Eugster (Zurich) for generous gifts of compounds, and theircontinued interest in this research. Correspondence toP.J.R., please.

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(Received April 27, 1983.Revised June 15, 1983.)