European Journal o f Pharmacology, 56 (1979) 287--290

287

© Elsevier/North-Holland Biomedical Press Short communication C O O P E R A T I V E I N T E R A C T I O N S A T [3H] K A I N I C A C I D B I N D I N G S I T E S I N R A T A N D HUMAN CEREBELLUM EDYTHE D. LONDON and JOSEPH T. COYLE * Departments o f Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School o f Medicine, Baltimore, Maryland 21205, U.S.A.

Received 24 April 1979, accepted 25 April 1979

E.D. LONDIN and J.T. COYLE, Cooperative interactions at [3H]kainic acid binding sites in rat and human cerebellum, European J. Pharmacol. 56 (1979) 287--290. Inhibition of the specific binding of [3H]kainic acid was studied in membranes isolated from rat and human cerebellum; the sequence of potencies in both species were: kainic acid > L-glutamic acid > dihydrokainic acid > D-glutamic acid. Whereas the Hill coefficient for unlabelled Kainate was 1.0, dihydrokainic acid and D- and L-glutamic acids exhibited negative cooperativity with Hill coefficients of near 0.5. This allosteric interaction of glutamic acid at the kainic acid recognition site suggests a biochemical correlate for the synergistic effects of these compounds in vivo. Dihydrokainie acid Cerebellum

Kainic acid

Glutamie acid

1. I n t r o d u c t i o n Kainic acid (Ka), a c o n f o r m a t i o n a l l y r e s t r i c t e d a n a l o g u e o f L - g l u t a m i c acid, is a p o t e n t n e u r o e x c i t a n t a n d n e u r o t o x i n . I t has b e e n suggested t h a t t h e n e u r o t o x i c a c t i o n o f kainic acid results f r o m its excessive activation of excitatory glutamate receptors located o n v u l n e r a b l e n e u r o n s ( O l n e y et al., 1974). R e c e n t ligand b i n d i n g studies h a v e i d e n t i f i e d r e c e p t o r sites t h a t a p p e a r to m e d i a t e the e f f e c t s o f kainic acid ( L o n d o n a n d C o y l e , 1 9 7 9 ) . In t h e p r e s e n t s t u d y , we have e x a m i n e d t h e kinetics o f i n h i b i t i o n o f D- a n d L-glutam a t e a n d d i h y d r o k a i n i c acid w i t h t h e b i n d i n g o f [ 3 H ] k a i n i c acid to r e c e p t o r sites in r a t a n d h u m a n c e r e b e l l u m to d e t e r m i n e h o w t h e s e

* To whom correspondence should be addressed: Joseph T. Coyle, M.D., Department of Pharmacology, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, U.S.A.

Cooperativity

Receptor binding

agents i n t e r a c t w i t h k a i n a t e r e c e p t o r s . T h e c e r e b e l l u m was selected b e c a u s e kainic acid is n e u r o t o x i c in this region ( H e r n d o n a n d C o y l e , 1977); m o r e o v e r , t h e c e r e b e l l u m contains o n l y o n e p o p u l a t i o n o f b i n d i n g sites f o r kainic acid w h e r e a s m o s t o t h e r brain regions possess at least t w o t y p e s o f r e c e p t o r sites ( L o n d o n and C o y l e , 1979), w h i c h w o u l d c o m p l i c a t e kinetic i n t e r p r e t a t i o n s .

2. Materials a n d m e t h o d s Male S p r a g u e - D a w l e y rats ( 1 5 0 - - 2 2 5 g ) w e r e sacrificed b y d e c a p i t a t i o n ; t h e i r cerebella w e r e dissected o u t , f r o z e n o n d r y ice a n d s t o r e d in a liquid n i t r o g e n freezer. H u m a n brain tissue ( 1 2 - - 1 4 h p o s t - m o r t e m ) was obt a i n e d either f r o m t h e Medical E x a m i n e r ' s O f f i c e o f t h e City o f B a l t i m o r e or t h e D e p a r t ment of Pathology, Johns Hopkins School of Medicine; cerebellar s a m p l e s w e r e similarly f r o z e n a n d s t o r e d until t h e t i m e o f assay.

288 Specific binding of [3H]kainic acid (4.1 Ci/mM and 2 . 6 C i / m M ; Amersham Searle; Arlington Heights, Ill.) to cerebellar membranes was measured as described previously (London and Coyle, 1979). Samples of cerebellum were sonified in 1 0 0 v o l of 50 mM Tris-citrate buffer, pH 7.1 at 2°C with a Sonifier Cell Disruptor (Model W 1 8 5 ; Heat Systems--Ultrasonics, Inc.; setting 4, 30 sec.). During the preparation and subsequent incubation, the homogenates were maintained at 2°C. The membranes were isolated b y centrifugation at 48 000 × g × 10 min. The membranes were subsequently suspended, washed extensively in distilled water and then resuspended in the same volume of the Tris-citrate buffer used in the original homogenization. To 13 ml Teflon centrifuge tubes, 1 ml portions of the membrane suspension were added; in some tubes, 200 nmoles of unlabelled kainic acid (Sigma Chemical Co.; St. Louis, MO.) was included for measurement of non-specific binding; in other tubes, various amounts of the potential inhibitors of specific binding [unlabelled kainic acid, L-glutamic acid (Sigma), D-glutamic acid (Sigma) and dihydrokainic acid (prepared by Drs. I. McDermott and C. Robinson of this Department of Pharmacology)] were added. The volume was brought to 1.9 ml with glass distilled water, and the assay was initiated by the addition of either 50 pmoles or 10 pmoles of [3H]kainic acid in 0.1 ml of water. Incubations were run for 60 min and terminated by centrifugation at 48 000 × g (10 min). The resultant pellets were rinsed superficially with water and then solubilized by incubating for 30 min at 55°C with 1 ml of Protosol (New England Nuclear Corp; Boston, MA). Specific binding was defined as the difference between total binding with radioligand alone and nonspecific binding measured in the presence of 100 ttM unlabelled kainic acid. The ICs0 (concentration producing 50% of inhibition of specific binding) values for the inhibitors were determined by log-probit analysis. The affinity of each inhibitor for [3H]KA binding sites (i.e., Ki) was obtained

E.D. LONDON, J.T. COYLE from the equation: Ki = ICs0/(1 + [KA]/KD) The K D for the specific binding [3H]kainic acid to cerebellar membranes for both rat and man is approximately 2.5 × 10 -s M (London and Coyle, 1979 and unpublished observations). Hill coefficients were calculated by linear regression of the line obtained by plotting the log of [% Bmax/100%--% Bmax ] versus the log of the concentration of inhibitor.

3. Results The inhibition dissociation constant (Ki's) for the four inhibitors of the specific binding of [3H]kainic acid in rat and human cerebellar membranes are shown in table 1. The Ki's for unlabelled kainic acid in rat (12 nM) and man (14 nM) are within the range of K D values obtained from saturation isotherms with [3H]kainic acid (London and Coyle, 1979). Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid reduces the affinity of molecule for receptor site 50-60fold. L-glutamic acid exhibits a 40-fold lower affinity for the rat receptor and 100-fold lower affinity for the human receptor. Both the rat and human receptor sites possess significant stereospecificity with D-glutamic acid having a 10- to 20-fold lower affinity than the L-isomer. Log-probit analysis of the inhibition curves for unlabelled kainic acid and L-glutamic acid for the specific binding of [3H]kainic acid to rat cerebellar membranes is shown in fig. 1. Whereas inhibition of specific binding from 10--90% t o o k place within two log units of kainic acid concentrations, the inhibition curves for L-glutamic acid extended over three log units of inhibitor concentration. Similarly, inhibition curves that extended over three log units were observed with L-glutamic acid in the human cerebellum and with dihydrokainic acid and D-glutamic acid in both the rat and human preparations. Hill

COOPERATIVITY AT KAINIC ACID RECEPTORS

289

TABLE 1 Affinities and Hill coefficients for [ 3H ]kainic acid binding sites in rat and human cerebellar membranes. Cerebellar membranes were incubated with 5 nM or 25 nM [3H]kainic acid in the presence of varying concentrations of the compounds listed below; ICs0 values were determined by log-probit analysis from which Kis were derived. Each experiment was performed at least twice. Hill coefficients were calculated by linear regression as the slope of log % Bmax vs. log concentration of inhibitor. Compound

Rat

Kainic acid L-Glutamic acid Dihydrokainic acid D-Glutamic acid

Man

K i (M)

Hill coefficient

r

K i (M)

Hill coefficient

r

1.2 5.0 6.7 1.0

0.96 0.62 0.53 0.63

0.85 0.90 0.88 0.94

1.4 1.5 7.4 9.4

0.92 0.52 0.47 0.43

0.99 0.92 0.88 0.95

x x x x

10 -s 10 -7 10 -7 10 -s

c o e f f i c i e n t s were c a l c u l a t e d for t h e displacem e n t curves f o r all f o u r c o m p o u n d s . I n b o t h rat and h u m a n cerebellar p r e p a r a t i o n s , the Hill coefficients for d i s p l a c e m e n t w i t h unlabelled kainic acid were near u n i t y ; h o w e v e r , t h e Hill c o e f f i c i e n t s c a l c u l a t e d f o r L-glutamic acid, D - g l u t a m i c a n d d i h y d r o k a i n i c acid were all a p p r o x i m a t e l y 0.5, w h i c h is indicative o f negative c o o p e r a t i v i t y .

95 90 70 50 30 I0

I

I 0 -9

I

I

I0 - 8

I0 -7

OI

I0 -6

I

I

I

I

I0 - 5

I0 - 4

I0 -3

10-2

Fig. 1. Inhibition of [3H]kainic acid binding by un-

labelled kainic acid and L-glutamic acid. Rat cerebellar membranes were incubated with 25 nM [3H]kainic acid in the presence of various concentrations of unlabelled kainic acid and L-glutamic acid. ICs0 values were determined by log-probit analysis. Ordinate: percent of maximal binding. Abscissa: concentration of inhibitor. (M).

x × x x

10 -8 10 -6 10 -7 10 -6

4. Discussion These studies d e m o n s t r a t e t h a t t h e inhibit i o n o f specific binding o f [3H]kainic acid t o r e c e p t o r s in rat a n d h u m a n c e r e b e l l u m b y Da n d L-glutamic acids does n o t f o l l o w simple mass a c t i o n kinetics as with unlabelled kainic acid. Whereas t h e d i s p l a c e m e n t o f [3H]kainic acid f r o m r e c e p t o r s with unlabelled kainic acid o c c u r s within t w o log units o f c o n c e n t r a t i o n , t h e i n h i b i t i o n b y D- a n d L-glutamic acid takes place over t h r e e log units. Hill plots o f these inhibition curves indicate t h a t kainic acid has a slope near u n i t y whereas t h e D- and L-glutamic acids exhibit slopes o f approxim a t e l y 0.5. A n Hill p l o t slope o f 0.5 is c o m patible with negative c o o p e r a t i v i t y . F u r t h e r m o r e , r e d u c t i o n o f t h e i s o p r o p y l e n e side chain o f kainic acid to f o r m d i h y d r o k a i n i c acid results in a derivative t h a t n o t o n l y has a lower a f f i n i t y f o r the r e c e p t o r b u t also exhibits negative c o o p e r a t i v i t y similar t o Da n d L - g l u t a m a t e . N o t a b l y , d i h y d r o k a i n i c acid lacks n e u r o e x c i t a t o r y a n d n e u r o t o x i c effects ( S c h w a r c z et al., 1978). This result indicates t h a t the d o u b l e b o n d in t h e side chain o f kainate, a s t r u c t u r e t h a t has n o h o m o l o g u e in t h e g l u t a m a t e m o l e c u l e , plays an essential role in t h e binding o f [3H]-kainic acid t o the receptor. Negative c o o p e r a t i v i t y implies an allosteric i n t e r a c t i o n o f g l u t a m a t e at the kainic r e c e p t o r

290 site. It is unlikely that this represents an interaction at shared sites on the same r e c e pt o r since kainic acid is a poor inhibitor (ICs0 1 mM) at the specific binding site for L [3H]glutamic acid in rat cerebellar membranes (Foster and Roberts, 1978). Such an allosteric interaction between glutamic acid and kainic acid is consistent with several aspects of kainate's action. R ecent electrophysiologic studies indicate that kainic acid and glutamic acid act at separate receptors sites in mammalian CNS (Hall et al., 1978). At the crustacean neuromuscular junction, kainic acid is a weak excitant but potentiates the depolarization induced by glutamate; and it has been suggested that kainic acid may activate extrajunctional receptors at this glutamatergic synapse (Shinozaki and Shibuya, 1974). Kainic acid has been shown to be a p o t e n t and selective n e u r o t o x i n when injected into the striatum (Coyle and Schwarcz, 1976). T h e acute neurochemical alterations following striatal injection of kainic acid can be reproduced in slices incubated in vitro only when kainic acid is incubated in c om bi nat i on with L-glutamic acid (Biziere and Coyle, 1978b). F u r t h e r m o r e , cortical ablation, which destroys a major glutamatergic projection to the striatum, markedly attenuates the neurotoxic action of kainic acid in the striatum (Biziere and Coyle, 1978a). T ha t glutametergic striatal afferents play a permissive role in the n e u r o t o x i c action of kainic acid is additionally supported by the fact that coadministration of kainic acid and glutamic acid in the decorticate striatum resulted in partial restoration o f kainic acid's neurotoxic action (Biziere and Coyle, 1978a). Thus, negative cooperativity of glutamate's interaction at the recognition site for [3H]kainic acid provides a biochemical correlate for the synergistic interactions between these agents demonstrated in neurophysiological and neurotoxicological studies. Taken together, the anomalous kinetics of displacement of [3H]kainic acid f r om its r e c e p t o r sites by glutamic acid and by the inactive derivative

E.D. LONDON, J.T. COYLE dihydrokainic acid furt her erode the concept that kainic acid is simply a p o t e n t and specific glutamate r e c e p t o r agonist.

Acknowledgements

This work was supported by USPHS grants MH 26654, NS13584, RCDA Type II MH00125 and grants from the National Foundation and McKnight Foundation to JTC and USPHS Fellowship MH 07142-01 to EDL. We thank Gary Schattschneider for excellent technical assistance and Princie Campbell and Carol Kenyon for secretarial help. We are

also grateful to Dr. Morley Hollenberg for his comments.

References

Biziere, K. and J.T. Coyle, 1978a, Influence of cortico-striatal afferents on striatal kainic acid neurotoxicity, Neurosci. Lett. 8,303. Biziere, K. and J.T. Coyle, 1978b, Effects of kainic acid on ion distribution and ATP levels of striatal slices incubated in vitro, J. Neurochem. 31,513. Coyle, J.T. and R. Schwarcz, 1976, Model for Huntington's chorea: lesion of striatal neurons with kainic acid, Nature 2 6 3 , 2 4 4 .

Foster, A.C. and P.J. Roberts, 1978, High affinity L-3H-glutamate binding to post-synaptic receptor sites on rat cerebellum membranes, J. Neurochem.

31, 1467. Hall, J.G., T.P. Hicks and H. McLennan, 1978, Kainic acid and the glutamate receptor, Neurosci. Lett. 8,171. Herndon, R. and J.T. Coyle, 1977, Selective destruction of neurons by a transmitter agonist, Science

198, 71. London, E.D. and J.T. Coyle, 1979, Specific binding of [3H]Kainic acid to receptor sites in rat brain, Mol. Pharmacol. (in press). Olney, J.W., V. Rhee and O.L. Ho, 1974, Kainic acid:

a powerful neurotoxic analogue of glutamate, Brain Res. 77,507. Schwarcz, R., Scholz, D. and Coyle, J.T. 1978, Structure-activity relations for the neurotoxicity of kainic acid derivatives and glutamate analogues, Neuropharmacol. 1 7 , 1 4 5 . Shinozaki, H. and I. Shibuya, 1974, Potentiation of glutamate-induced depolarization by kainic acid in the crayfish opener muscle, Neuropharmacol. 13,

1057.