~ Pergamon

0742-s413(94)E0006-u

Comp. Biochem. Physiol. Vol. I08C, No. 2, pp. 205-214, 1994 Elsevier Science Ltd Printed in Great Britain

0742-8413/94 $7.00 + 0.00

An endogenous ligand for the kainate-type binding sites from rat brain

Paolo Migani,* Elisabetta CianiJ" Marco Virgili " and Ottavio Barnabeit *Faculty Center for Biological Sciences, University of Ancona, Via Brecce Bianche, 60100 Ancona, Italy; and ~'Department of Biology, University of Bologna, Via Belmeloro 8, 40126 Bologna, Italy

Extracts from the rat brain were screened to identify a putative endogenous ligand for the binding sites of the neuroexcitant kainic acid (KA). The extracted substances were separated by chromatographic techniques and tested for their ability to inhibit KA binding to fish synaptosomes and to membranes from rat brain. A substance isolated in this way (rat kainate-binding inhibitor, RKBI) displays a competitive interaction with KA for the low-affinity binding sites in rat brain membranes. According to the separation behavior in the purification step, RKBI is distinct from an inhibitor formerly isolated from fish nervous tissue (KBI). The substance exhibits positive co-operativity with KA for a very-low-affinity site population, particularly concentrated in the cerebellum, and could play a physiological role in this area.

Key words: Kainic acid; Binding sites; Rattus norvegicus; Carassius auratus.

Comp. Biochem. Physiol. 108C, 205-214, 1994.

Introduction Kainic acid, a well-known neuroexcitant and neurotoxin, binds to receptor sites in the nervous tissue which belong to the general group of membrane receptors for excitatory amino acids (EAA) (Watkins and Evans, 1981; Foster and Fagg, 1984). Among the EAA sites, a distinct group binds N-methyl-D-aspartate (NMDA) while others bind kainic acid (KA) and

-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and are not easily distinguishable from one other (Cotman

Correspondence to: P. Migani, Centro di Spesa della Facolt~i di Scienze dell'Universit~ di Ancona, Via Brecce Bianche, 60100 Ancona, Italy. Tel. 071- 2204515; Fax 071-2204513.

Received 23 November 1993; accepted 8 March 1994.

and Iversen, 1987; Henley and Barnard, 1991).

The physiological role of the kainate-type EAA receptors is not yet well understood: this is partially due to the fact that different sub-types exist which are distinguishable by their binding kinetics or by their structural characteristics, elucidated by molecular biology techniques (Barnard and Henley, 1990).

In the mammalian brain, two site popu- lations bind KA with high and low affinity (the KD being in the nanomolar and tens of nanomolars range) (Foster and Fagg, 1984); these sites should correspond to sev- eral monomeric receptor proteins recently identified from different DNA clones (Boulter et al., 1990; Egebjerg et al., 1991;

205

206 Paolo Migani et al.

Bettler et al., 1992). Another monomeric kainate binding protein (KBP) has been isolated from the chick cerebellum (Gregor et al., 1988); a similar protein (kainic acid receptor, KAR) has been purified from the frog brain (Hampson and Wenthold, 1988).

It is generally believed that the physio- logical ligand for the kainate-type sites (as well as for other EAA sites) is glutamate, since this substance is the most widespread among the endogenous compounds recog- nized by the sites. This idea can be chal- lenged on the basis of the heterogeneity of the kainate sites, which is compatible with the existence of more than one physio- logical ligand: accordingly, a recent report describes a site in the fish brain whose binding to KA is poorly sensitive to the displacing action of glutamate (Davis et al., 1992).

One of us has previously reported (Mi- gani, 1990) the isolation of a low molecular weight kainate-binding inhibitor (KBI) from the nervous tissue of the goldfish, a species with a particularly high density of KA sites (Migani et al., 1985; Henley and Oswald, 1988): it was suggested that this molecule (or a structurally similar sub- stance) can be regarded as a putative en- dogenous ligand of the KA sites in the fish brain and in the avian cerebellum.

We undertook the present research to check the possibility that a similar sub- stance exists in mammalian brain. We have adopted extraction and purification pro- cedures for the rat brain tissue similar to those formerly used for the fish tissue. To check for the presence of KA-displacing substances in the chromatographic frac- tions from the various purification steps we have tested their potency on the (3H)KA binding on goldfish spinal cord synapto- somes, assuming that the putative rat ligand could be also active as displacer in fish sites. The synaptosomal fraction from fish nervous tissue is more easily and rapidly prepared than the mammalian frac- tion (commonly used for binding assays) and assures a better sensitivity to the bind- ing test, due to the higher density of KA sites (Migani et al., 1985). An active sub- stance isolated in this way (rat kainate binding inhibitor, RKBI) was subsequently tested on KA binding in rat brain mem- branes.

Materials and Methods Extraction and purification of R K B I

A typical extraction-purification exper- iment involved the use of the entire brain (forebrain and cerebellum) from three to four adult rats (Rattus norvegicus, Wistar strain, 150-200g in weight). The brains were chilled in a Petri dish on ice and stored at -80°C for a period ranging from 1 day to 1 month. The tissue was then weighed (5-8 g) and homogenized in a laboratory grinder with rotating blades (about 8000rpm, 3min) with 15vol 75% (v/v) ethanol at about -15°C. The homogenate was centrifuged (3000g, 30 min, 4°C); the pellet was homogenized using the same procedure and again centrifuged. The supernatants were pooled and the bulk of the solvent was removed by a rotating evaporator; the residue was suspended in 0.1 M formic acid (30 ml) and the acid-in- soluble material was removed by centrifu- gation (3000 g, 15 min, 4°C). The pellet was washed by resuspension in 5 ml of the same solvent and again centrifuged (twice). The supernatant was freeze-dried; the dried ma- terial was resuspended in 4 ml 0.1 M formic acid and added to 1 ml of a suspension (10%, w/v) of the same polyacrylamide gel used for the following gel-permeation step. After a few minutes on ice, a centrifugation step (3000g, 15min, 4°C) yielded a clear supernatant, which was freeze-dried. The dried material was then applied to a 1.6 x 67cm polyacrylamide gel column (Bio-Gel P2; Bio-Rad, Richmond, CA) and eluted with 0.1 M formic acid, at a flow-rate of 0.125ml/min (gel-permeation step). A trace amount of 14C-labelled glutamate (0.5/iCi; 50 mCi/mmole; New England Nu- clear, Boston, MA) was introduced in the samples to mark the endogenous glutamate. Fractions were collected every 40min (5.0 ml); aliquots of 50/tl were utilized to detect the position of glutamate, by liquid scintillation counting. Other 125/al aliquots were rapidly dried in a rotating concentra- tor, resuspended in the same volume of Ringer saline and used to detect the presence of KA displacing substance (see below).

The active fractions different from those containing glutamate were pooled, freeze- dried, resuspended in 10 mM formic acid

A mammalian kainate-binding inhibitor 207

and injected into a high-performance reverse-phase chromatographic system (HPLC, step I). The system utilized a 1.8 x 25 cm C18 column (particle size 5 #m; Supelcosil; Supelco, Bellefonte, PA) which was eluted at 3.0ml/min with 10mM formic acid (medium A) and linearly- increasing amounts of methanol, up to the 30% (v/v) (medium B). One-minute frac- tions were collected. To mark the position of glutamate the ]4C-labelled compound was detected in 50/~1 aliquots; other 70/~1 aliquots were dried, resuspended in 125 pl Ringer saline and used to detect the KA- displacing substances. The active fractions were pooled, dried and resuspended in the starting medium of the second, ion-pair, HPLC step. This step used the above- described C18 column, eluted with 0.075% (v/v) triethylamine buffered to pH 6.0, with formic acid (medium A) at a flow-rate of 3.0 ml/min: after 10 min, a linear gradi- ent of methanol was started, up to the 20% in volume (medium B). One-minute frac- tions were collected and tested for the dis-

placing activity as described for the first step.

The same procedure was used for the brain of 5-12 animals per group from post- natal days 1-18 in age-related measure- ments; the same procedure was also used in some experiments with the brain-spinal cord tissue from 30 goldfish (7-12cm in length).

The presence of binding-inhibiting sub- stances in the chromatographic fractions was tested on the (3H)KA binding in fish spinal cord synaptosomes, as previously described (Migani et al., 1985). Briefly, spinal cords from two to three goldfish (Carassius auratus, 7-10 cm in length) were homogenized in 10% (w/v) sucrose; the synaptosomal fraction was isolated by differential centrifugation and suspended in Ringer saline to reach a protein content of about 0.4 mg/ml. Fifty-microlitre aliquots of this suspension were incubated with 50 #1 of the saline-reconstituted chromato- graphic fraction and with 10riM (3H)KA (30-50 ci/mmole; Amersham, Bucks, U.K.)

.<

v

2.0

1.6

1.2

0.8

0.4

F 1 J I

r L

J , i I . I I

tO 20

Fraction no.

~ - K B I - - ~

1.0 ~ ,.,

o,s ~._.~ 0.6

o., 0.2

0.2 ~ _ ~

o.1 , ~ 2 ~

i i i

Fig. 1. Purification of RKBI from extracts of rat brain. Step 1: gel permeation. The substances extracted from the brain tissue were separated by elution from a polyacrylamide gel column, as described in the Materials and Methods section. A typical chromatogram, representing the optical density of the eluates, is presented (solid line). The level of radioactivity corresponding to (14C)glutamate (used as tracer of the endogenous glutamate) is represented by the broken line. The step-wise line (upper side of the figure) shows the inhibitory effect of the chromatographic fractions on the KA binding on fish spinal cord synaptosomes: the line represents the mean values (+ SEMs, bars) of duplicate measurements from nine experiments. The arrow marks the fraction corresponding to the total volume (Vr) of the gel column. The horizontal bar marks the fractions pooled for the next purification step. The area labelled "KBI" marks the position of a peak of inhibitory activity detected in experiments where

goldfish nervous tissue was used as starting material.

CBPC 108/2--F

208 Paolo Migani et al.

to a final volume of 150/~1 in plastic micro- tubes (0.4 ml). After a 45-min period on ice the synaptosomal fraction was pelleted by centrifugation (10,000g, 15min, 4°C) and the supernatant was carefully aspi- rated; the pellet was dissolved by 20/~1 tissue-solubilizer (Soluene; Packard, Down- ers Grove, IL); 300/~1 scintillation mixture (Pico-fluor 40; Packard) were added to the tubes and the bound radioactivity was measured by liquid scintillation spec- trometry.

The amounts of RKBI in the HPLC fractions were quantified by comparing their inhibitory activity with that of serially- diluted non-labelled KA, after linear log- probit t ransformation of the displacement curve. One kainate-like unit (KLU) was defined as the amount of RKBI with the same potency of 1 nmole of KA on the fish synaptosomal binding. It was assessed, in this way, that the recovery of activity from the first to the second HPLC step was always more than 80%.

Bind ing m e a s u r e m e n t s on ra t brain m e m - b ranes

To test the effect of RKBI on rat mem- branes, these were prepared from the synaptosomal fraction from the entire brain of adult animals by a reported pro- cedure (Nieto-Sampedro et al., 1980) which includes osmotic shock and repeated freeze-thaw steps. The membranes were extensively washed with water and finally resuspended in 50 mM Tris-HC1 buffer (pH 7.2) to a protein content of about 5 mg/ml.

Fifteen-microlitre aliquots of the suspen- sion were used for the binding assay in a micro-system (45/~1 final volume) in plastic Eppendorf-type (1.5 ml) tubes, with (3H)KA (30-50 Ci/mmole, Amersham) and RKBI from the last purification step, in Tris buffer. After a 90-min incubation on ice, the tubes were centrifuged (10,000g, 15 min, 4°C), the supernatants were aspi- rated, the pellets were rapidly washed with 1 ml of ice-cooled Tris-buffer and dissolved

0.8

0.6 Q

~o o.4

K B I

I I I I

0 5 10 15

Time (min)

!

I

Inj.

4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

<

0.9 ~ : ~

0 7

0.5 ~.~

0.3 I ~ O.l ~

0.75 ~ ~

0.25 ~ "~

100

E 60

20 N

i

i

Fig. 2. Purification of RKBI. Step 2: reverse-phase HPLC. The binding-inhibiting material from the preceding (gel permeation) step was fractionated by reverse-phase high-performance chromatography, as detailed in Materials and Methods: a typical chromatogram is presented. The dotted line indicates the increment of methanol in the elution medium; the broken line refers to the amounts of the tracing (14C)-glutamate in the chromatographic fractions. The upper line represents the binding-inhibition test for the chromatographic fractions and shows the mean values (+ SEM) of binding measurements from nine experiments (in duplicate). The active fractions (bar) were used for the next purification step. The peak of inhibitory activity

shifted to the position marked by "KBI" when samples from goldfish tissue were used.

A mammalian kainate-binding inhibitor 209

in 100 #1 0.2% (w/v) sodium dodecyl sul- phate (SDS) in 0.5 M NaOH. One millilitre of liquid scintillation mixture (Pico-fluor 40; Packard) was added to the tubes and they were counted for radioactivity.

For dose-related binding measurements the specific activity of (3H)KA was lowered to one-tenth of the original with the un- labelled compound. The non-specific bind- ing was evaluated by duplicate samples with 0.1mM KA.

The protein content of the fish synapto- somal and rat membrane suspension, as well as that of the pellet of the binding tests, was measured after dissolution in SDS-NaOH, by the Lowry et al. (1951) method. The amino acid composition of the gel-permeation fractions was evaluated in one case by a reported HPLC procedure (Hill et al., 1979), using the O-phthalalde- hyde (OPA) derivatization.

Results

The substances extracted by ethanol from the rat brain tissue were first separated by gel-permeation on a polyacrylamide column, a step which produced the chro- matogram in Fig. 1. The KA-displacing test on the fractions led to identification of two

areas of activity: the first and major one corresponds to the position of glutamate, as pointed out by the position of the ~4C- labelled compound. Other displacing sub- stances such as aspartate and glutamine share this position with glutamate, as testified by serial amino acid analysis of the fractions (not shown).

The second and smaller area (horizontal bar) corresponds to an active substance (or substances) whose migration on the column was retarded by interaction with the gel since its position corresponds to a volume of the elution medium exceeding the total volume of the column (VT). It is noteworthy that this area is partially superimposed on that of fish KBI activity, eluted and de- tected by the same system. In the second purification step, the substances of this area were subjected to reverse-phase HPLC, which led to the chromatogram in Fig. 2. Apart from the activity of the residual glutamate, marked by the bulk of the tracer compound, a single peak of displacement is detectable (bar). Intriguingly, a small portion of radioactivity introduced as (~4C)glutamate was found in this position but, by comparison with the preceding peak, the main displacing activity should not be due to authentic glutamate but

0.7

0.5

0.3

<

T 0.1

Inj. t O 0 D q

l ~ O O e O O 0 ~ O I ~ O I ~ Q

< ~.o~ z

o.s ~ ~ o.6 . ~ . ~

0.4 ,-, .~

0.2 [ ~

loo E

o

60

20

I I I } | I 0 5 10 15 20 25

Time (rain)

Fig. 3. Purification of RKBI. Step 3: ion-pair HPLC. The active substances (bar) were detected as explained in the Materials and Methods section and in the legends of the precceding figures. The dotted line shows the gradient of methanol in the elution medium.

210 Paolo Migani e t al.

rather to a different substance (or sub- stances). In the present report, the displac- ing activity will be referred to a single rat kainate-binding inhibitor, or RKBI. The position of this active substance in the chromatogram precedes that of the main peak of the KBI activity so that the sub- stance itself should be less hydrophobic than the compound extracted from the fish tissue. Furthermore, the substance is retained longer than KBI by the column in the ion-pair H P L C of the third purifi- cation step (Fig. 3): in agreement with a current model of the ion-pair chromatog- raphy, considered as a weak ion-exchange system (Deming, 1985), this result should mean that RKBI is more acidic than fish KBI.

The amount of purified substance per tissue weight unit was found to be fairly constant in different postnatal ages (Fig. 4a); the recovered activity was significantly correlated with the amounts of extracted tissue (Fig. 4b). Measurements of the specific K A binding on washed membranes from the rat brain led to the Scatchard plot (Scatchard, 1949) of Fig. 5 showing two site populations with different affinities for the ligand, corresponding to the low-affinity populat ion described in the literature (Foster and Fagg, 1984) and to a very-low- affinity one. N o at tempt was made to detect the high-affinity site populat ion described in the literature with the ligand at concen- trations in the nanomolar range. The ad- dition of a fixed amount of RKBI (lower plot) completely abolished the port ion of the graph corresponding to the very-low- affinity sites. For the low-affinity sites the inhibition was less prominent and did not significantly affect the site density par- ameter (Bmax), thus demonstrating that the action of R K B I was purely competitive. These results show a different sensitivity of the low- and very-low-affinity K A sites to RKBI: this is also shown by dose-related displacement experiments (Fig. 6), carried out at two K A concentrations to mark preferentially one of the two populations. The apparent half-maximum displacing concentration (IC50) is about one order of magnitude lower for the very-low-affinity sites than for the low-affinity ones. It is also noteworthy that the K A - R K B I inter- action with the very-low-affinity sites

0.3

0.2

,.d

O.11

(a)

• I I I

I I I I I I I ' J ' !I I

2 6 10 14 18 Adult

Postnatal age (days)

3.0

~d 2.5

a~ • ~ 2.0

"" 1.5 e ~

1.o

0.5 <

(b)

i / i I I i i i i I I

2 ; 6 8 10 12

Tissue weight (g)

Fig. 4. (a) Brain content of RKBI during postnatal development. RKBI was extracted from rat brain tissue at different developmental stages and purified as shown in the preceeding figures. The amounts of inhibitor were quantified by comparing their binding- displacing potency with that of KA, as detailed in the Materials and Methods section: one kainate-like unit (KLU) was defined as the amount of RKBI with the same potency of 1 nmol KA. The points on the figure represent the amount recovered after the first HPLC step, expressed per weight-unit of the extracted tissue, as mean _ SEM of the values from 4 to 6 separate experiments (values from the postnatal days l and l0 are from single experiments). Differences among the values of different stages were subjected to an analysis of variance (one-way, ANOVA) and were found to be not significant. (b) Correlation between the amount of RKBI and the weight of the extracted tissue. The points refer to the values of single experiments, where the extracted tissue was either from adult rats or animals at different development stages; values rep- resent the amount collected after the first HPLC purification step. The line was best-fitted to the points by the least-square method (r = 0.708) and the signifi- cance of its slope was checked by the Student's t-test

(P < 0.0001).

shows a degree of positive co-operativity demonstrated by the occurrence of binding values higher than that measured with K A alone, and by the slope of the Hill graph (Hill, 1910) (Hill number higher than 1.0).

A mammalian kainate-binding inhibitor 211

20

15

,o <

o c o n t r o l

• . R K B I ( ! K L U / r n ) )

0.2 0.6 1.0 1.4 1.8 2.2 Bound 3HKA (pmoles/mg protein)

!

2.6

Fig. 5. Dose-related binding of KA on rat brain membranes with and without RKBI: Scatchard Plot. The specific (total minus non-specific) binding of KA on rat brain membranes was measured at concentrations of the ligand from 5 to 1000 nM and data were expressed according to Scatchard (1949); points refer to the mean values of five separate experiments, in duplicate. According to a two-site model, the points corresponding to the control (-RKBI) measurements were fitted to a two-line graph by the least-square method (r > 0.98); the significance of the line parameters was checked by the Student's t-test (P < 0.001). The binding parameters corresponding to the site density and affinity constant were: 1. high-affinity sites, Bmx = 0.608 + 0.036 pmol/mg protein, KD = 34 + 3 nM; 2. low-affinity sites, Bm~x = 2.36 + 0.15 pmol/mg protein, KD ---- 366 + 4 nM. Points corresponding to measurements with RKBI were fitted to a single line (single site population) (P < 0.001, Student's t-test); the density parameter (Bm~ = 0.543 + 0.037 pmol/mg protein) was not significantly different from

that of high-affinity sites in the control graph (P > 0.2., Student's t-test).

D i s c u s s i o n

The present results demonstrate the exist- ence in the rat brain of a substance (or structurally related substances) different from glutamate which can bind to the kainate-type receptor sites, both on mem- branes from the same species and on fish synaptosomes. Due to the limited amount, no systematic research has been performed on the chemical nature of the kainate-bind- ing inhibitor (RKBI). No material was vis- ible after concentration and drying of the active fraction from the last purification step so that weighing by common labora- tory tools was not feasible. It is not possible to infer the molecular weight of RKBI from gel chromatography since the elution time was probably not correlated with the mol- ecular size, due to an interaction of the substance with the gel matrix which recalls that of the fish inhibitor KBI (Migani,

1990). Possibly, they share some structural features but the substance described here should be more acidic and less hydrophobic than the KBI, according to the HPLC data. Limited experiments by mass spectrometry (not shown) gave no significant structural data, probably due to the presence of inter- fering substances in the RKBI samples ob- tained from the last purification step. The existence of substances which co-eluted with RKBI is also supported by the fact that no correlation was found between the RKBI-displacing activity and the area of the corresponding peak of optical activity (chromatogram, Fig. 3).

The apparent concentration of RKBI in the brain tissue (about 0.2 KLU/g) is in the same order of magnitude of that of KBI (about 0.4 KLU/g) (Migani, 1990) and this parameter does not show major variations during postnatal development. This fact does not match the marked increase in the

212 Paolo Migani e t al.

"O

%. ¢.)

e~ o

k

1.0

0.6

0.2

100

(a) [KA] = 10 nM

~ "~ -1 ~/l~nH -- 1.1o

~ ~ L o g [RKBI]

I I q ¢ I .

101 102 103 104 [RKBI] (KLU/!)

Y

0.2

0.6

1.0

<

rq

"d g

I-,

1.4

1.2

1.0

0.8

0.6

0.4

0.2

100

(h) [KA] = 200 nM I

101 1 0 2 1 0 3

[RKBI] (KLU/I)

~ = 1 . 7 4 I | !

2 3 4 Log [RKBI]

I 1 0 4

0.2

0.6

1.0

Fig. 6. Displacement by RKBI of KA binding on rat brain membranes. Serially-diluted RKBI was tested at two different concentrations of labelled KA. Points and bars in (a) refer to the mean values + SEM of the specific binding data collected in five experiments, in which all the displayed concentrations were tested. The Hill (1910) graph (insert) was plotted from the mean values of the displaced fraction (Y) of the specific binding: only the values corresponding to the decreasing side of the displacement curve were used. Points in (b) refer to the mean values of binding data collected in three experiments with all the displayed concentrations of RKBI. For the Hill graph, the displacement from the maximum measured binding value was used. Lines in the Hill graph were fitted to the points by the least-square method (r > 0.98); the significance of the slope parameter was checked by the Student's t-test (P < 0.01). The arrows

point the RKBI concentrations of apparent half-maximum displacement (IC50).

K A sites which occurs in some rat brain areas from the first to the third postnatal week (Erdo and Wolff , 1990; Miller et al., 1990). Assuming the existence o f a relation- ship between RKBI content and K A site density, it must be noted, however, that other rat brain areas undergo a less pro- nounced increase in the latter parameter or even a decrease (hippocampal CA1, thalamic regions) (Miller et al., 1990). Ac- cordingly, the mean value o f both par-

ameters could remain stable during development.

An interesting finding o f the present re- search concerns the different sensitivity o f the low- and very-low-affinity K A sites for the RKBI. Admitt ing that the binding inhi- bition is competit ive for the very-low- affinity sites as well as for the low-affinity ones, this difference should reflect a differ- ent affinity for the RKBI which should be the reverse o f that for KA. It must be noted

A mammalian kainate-binding inhibitor 213

that, in our hands, whole rat brain mem- branes displayed a KA site population with very low affinity (KD in the hundreds of nanomolar range) which has not been de- scribed in early reports on mammalian membranes (Foster and Fagg, 1984). Con- trol experiments (not shown) demonstrate that this population is particularly enriched in the cerebellum and scarce in the fore- brain and, therefore, should not be due to artifacts in binding measurement method- ology. A tentative explanation for the pre- vious failure to detect these sites could assume a dependence of their binding ac- tivity on C1 - ions which were omitted from the test solutions; it should be noted, how- ever, that a very-low-affinity kainate bind- ing site population has also been recently detected in a membrane fraction from the rat cerebellum by Viennot et al. (1991) in the absence of chloride ions. This popu- lation could be similar to the very-low- affinity site population present in high density in the pigeon (Henke et al., 1981) and fish cerebella (Ziegra et al., 1990; Tong et al., 1992) and could also correspond to the chick kainate-binding protein (KBP) (Gregor et al., 1988). It is noteworthy that RKBI exhibits a co-operative behaviour with KA for this site population, which resembles that of KA itself for pigeon (Henke et al., 1981), frog (Hampson and Wenthold, 1988) and fish (Migani et al., 1985) sites as well as that of KBI (Migani, 1990). This behaviour could represent the interaction of different receptors, carrying distinct sites for KA and RKBI. A similar interaction has been proposed elsewhere for cloned ionic channels activated by gluta- mate and kainate, whose gated conductance could be dependent on the positive inter- action between different monomeric sub- units (Boulter et al., 1990; Sakimura et al., 1990).

Due to the lack of structural data we cannot speculate on the chemical nature of RKBI, neither can we rule out the possi- bility that its displacing action is unspecific (mass-driven): the differential effect on the two binding site populations, however, does not support this possibility. We have not explored the action of RKBI towards the binding of other EAA ligands such as N M D A and AMPA: this can be a subject for future research which could produce

evidence on the problem of the site identity. Other research should explore the physio- logical role of this substance: due to the peculiar interaction of RKBI with the very- low-affinity KA binding sites in the cerebel- lum, this area seems particularly suitable for this research.

From a comparative point of view, it is noteworthy that a function associated with the very-low-affinity sites seems present in different areas of the fish brain, according to the site distribution (Tong et al., 1992; Ziegra et al., 1990) while it seems segregated in the cerebellum of birds and mammals (Henke et al., 1981); this fact could have significance for neural evolution and could be related to the structural differences of KBI and RKBI.

Acknowledgements--This work was supported by a grant from the Italian Ministry for University and Scientific Research (MURST). We thank Dr F. Giorgi of the Department of Biology, University of Bologna, for helpful assistance in line fitting.

References Barnard E. A. and Henley J. M. (1990) The non-

NMDA receptors: types, protein structure and molecular biology. Trends Pharmac. Sci. 11, 500-507.

Bettler B., Egebjerg J., Sharma G., Pecht G., Her- mans-Borgmeyer I., Moll C., Stevens C. F. and Heinemann S. (1992) Cloning of a putative gluta- mate receptor: a low-affinity kainate-binding sub- unit. Neuron 8, 257-265.

Boulter J., Hollman M., O'Shea-Greenfield A., Hart- ley M., Deneris E., Maron C. and Heinemann S. (1990) Molecular cloning and functional ex- pression of glutamate receptor subunit genes. Sci- ence 249, 1033-1037.

Cotman C. W. and Iversen L. L. (1987) Excitatory amino acid in the brain-focus on NMDA recep- tors. Trends Neurosci. 10, 263-265.

Davis R. E., Wilmot G. R. and Jang-Ho J. Cha (1992) Glutamic acid-insensitive [3H]kainic acid binding in goldfish brain. Brain Res. 571, 73-78.

Deming S. N. (1985) An ion-interaction model. In Handbook o f HPLC for the Separation o f Amino Acid, Peptides and Proteins (Edited by Hancock W. S.), Vol. 1, pp. 141-152. CRC Press, Boca Raton, FL.

Egebjerg J., Bettler B., Hermans-Borgmeyer I. and Heinemann S. (1991) Cloning of a eDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature 351, 745-748.

Erdo S. L. and Wolff J. (1990) Postnatal development of the excitatory amino acid system in visual cortex of the rat. Changes in ligand binding to NMDA, quisqualate and kainate receptors. Int. J. DevL Neurosci. 8, 199-204.

214 PaoloMigani et al.

Foster A. C. and Fagg G. E. (1984) Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors. Brain Res. Rev. 7, 103-164.

Gregor P., Eshlhar N., Ortega A. and Teichberg V. I. (1988) Isolation, immunochemical characterization and localization of the kainate sub-class of gluta- mate receptor from chick cerebellum. EMBO J. 7, 2673-2679.

Hampson D. R. and Wenthold R. J. (1988) A kainic acid receptor from frog brain purified using do- moic acid affinity chromatography. J. biol. Chem. 263, 2500-2505.

Henke H., Beaudet A. and Cu6nod M. (1981) Auto- radiographic localization of specific kainic acid binding sites in pigeon and rat cerebellum. Brain Res. 219, 95-105.

Henley J. M. and Barnard E. A. (1991) Comparison of solubilized kainate and ~-amino-3-hydroxy-5- methylysoxazolepropionate binding sites in chick cerebellum. J. Neurochem. 56, 702-705.

Henley J. M. and Oswald R. E. (1988) Solubilization and characterization of kainate receptors from gold- fish brain. Biochim. biophys. Acta 937, 102-111.

Hill A. W. (1910) The possible effects of the aggrega- tion of the molecules of hemoglobin on its dis- sociation curves. J. Physiol., Lond. 40, iv-vii.

Hill D. W., Walters F. H., Wilson T. D. and Stuart J. D. (1979) High-performance liquid chromato- graphic determination of amino acids in the pico- molar range. Analyt. Chem. 51, 1338-1341.

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.

Migani P. (1990) A competitive inhibitor of kainic acid binding from the goldfish nervous tissue. Brain Res. 518, 179-185.

Migani P., Virgili M., Contestabile A., Poli A., Villani L. and Barnabei O. (1985) [3H]Kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain. Brain Res. 361, 36-45.

Miller L. P., Johnson A. E., Gelhard R. E. and Insel T. R. (1990) The ontogeny of excitatory amino acid receptors in the rat forebrain--II. Kainic acid receptors. Neuroscience 35, 45-51.

Nieto-Sampedro M., Sheldon D. and Cotman C. W. (1980) Specific binding of kainic acid to purified subcellular fractions from rat brain. Neurochem. Res. 5, 591-604.

Sakimura K., Bujo H., Kushiya E., Araki K., Ya- mazaki M., Meguro H., Warashina A., Numa S. and Mishina M. (1990) Functional expression from cloned cDNAs of glutamate receptor species re- sponsive to kainate and quisqualate. FEBS Lett. 272, 73-80.

Scatchard G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672.

Tong C. K., Pan M. P. and Chang Y. C. (1992) Characterization of L-glutamate and kainate- binding sites in the brain of a freshwater fish, Tilapia monsambica. Neuroscience 49, 237-246.

Viennot F., de Barry J. and Bombos G. (1991) Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli. Neurochem. Res. 16, 435--442.

Watkins J. C. and Evans R. H. (1981) Excitatory amino acid transmitters. A. Rev. Pharmac. Toxicol. 21, 165-204.

Ziegra C. J., Oswald R. E. and Bass A. H. (1990) [3H]kainate localization in goldfish brain: receptor autoradiography and membrane binding. Brain Res. 527, 308-317.