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Binding of [³H] L-aspartate to membrane fractions of rat brain Stammers, Anthea Mary Tench 1982

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BINDING OF f 3H] L-ASPARTATE TO MEMBRANE FRACTIONS OF RAT BRAIN by ANTHEA MARY TENCH STAMMERS B.Sc , University of B r i t i s h Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Zoology Department, University of B r i t i s h Columbia) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1982 (C)Anthea Mary Tench Stammers, 1982 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e i i ABSTRACT The concerns of the present study were to determine 1) the conditions 3 necessary to measure displaceable [ H] L-aspartate binding to membrane f r a c t i o n s of the r a t brain, 2) whether the binding demonstrated the c h a r c t e r i s t i c s of the s i t e which i s active i n vivo, and 3) whether the a c i d i c amino acid neurotransmitters aspartate and glutamate bind to i d e n t i c a l or d i f f e r e n t s i t e s by comparing the pharmacological s p e c i f i c i t i e s 3 3 of the [ H] L-aspartate binding with that of [ H] L-glutamate. 3 The conditions of the [ H] L-aspartate binding assay were determined i n synaptosomal and t o t a l p a r t i c u l a t e f r a c t i o n s of whole rat brain. The reaction mixture which included the membrane f r a c t i o n suspended i n Tr i s - H C l buffer (pH 7.4) i n the presence or absence of the compound under t e s t , was incubated at 37°C f o r 30 minutes. The reaction was stopped by cen t r i f u g a t i o n and the r a d i o a c t i v i t y i n the p e l l e t counted by l i q u i d s c i n t i l l a t i o n spectrometry. 3 The [ H] L-aspartate binding was characterized i n t o t a l p a r t i c u l a t e f r a c t i o n s of rat cerebellum. The apparent d i s s o c i a t i o n constant (K^,) and maximum binding (Bmax), as determined by Scatchard a n a l y s i s , are 1.64 + 0.34 pM and 7711 + fmol/mg protein r e s p e c t i v e l y . The displaceable binding i s r e v e r s i b l e , saturable, independent of the presence of Na +, has an a f f i n i t y In the range where the neurotransmitter i s a c t i v e i n vivo, and demonstrates a pharmacological s p e c i f i c i t y which includes s t e r e o s p e c i f i c i t y . The compounds tested to demonstrate the pharmacological s p e c i f i c i t y were L-aspartate (IC^n = 1*81 VM), D-aspartate ( I C 5 0 = 46.6 uM), L-glutamate ( I C 5 Q = 1.24 uM), N-methyl-DL-aspartate ( i n a c t i v e ) , kainate ( i n a c t i v e ) , D-alpha-aminoadipate ( i n a c t i v e ) , and i i i L-alpha-aminoadipate (IC^Q = 7 . 1 2 yM). The pharmacological s p e c i f i c i t y 3 3 of [ H] L-aspartate binding was d i f f e r e n t from that of [ H] L-glutamate. When the binding data only are considered, therefore, separate receptors f o r aspartate and glutamate are i n d i c a t e d . 3 The pharmacological s p e c i f i c i t y of the [ H] L-aspartate binding, that i s the a f f i n i t y of the binding s i t e f o r N-methyl-DL-aspartate, D- and L-alpha-aminoadipate, however, does not c o r r e l a t e with the potency of these compounds derived from iontophoretic studies. L-alpha-aminoadipate i s very e f f e c t i v e while N-methyl-DL-aspartate and D-alpha-aminoadipate do not 3 d i s p l a c e the [ H] L-aspartate binding. In iontophoretic studies, N-methyl-D-aspartate and D-alpha-aminoadipate are very potent as compared to aspartate while L-alpha-aminoadipate Is i n a c t i v e . The 3 [ H] L-aspartate binding then may not represent the s i t e which Is active i n vivo. The c h a r a c t e r i s t i c s of the aspartate s i t e i n vivo, however, may not be t r u e l y represented i n iontophoretic studies because of, f o r example, uptake of the compounds. The aspartate binding s i t e , therefore, must be i d e n t i f i e d as that which i s activated i n v i v o . The question of separate receptors f o r aspartate and glutamate then must s t i l l be resolved. i v ACKNOWLEDGEMENTS I am g r a t e f u l to Dr. Hugh McLennan and Dr. John Steeves under whose guidance and support these studies were performed. I would also l i k e to thank Dr. Andrew Larder f o r his guidance and enjoyable companionship, Mrs. Yvonne Heap f o r her help with a l l the glassware, and Ms. Judy Smith f o r the f a n t a s t i c job she did i n typing t h i s t h e s i s . Thanks also to my husband, Michael, f o r h i s love and the help which provided me with extra time to work on t h i s t h e s i s . V TABLE OF CONTENTS CHAPTER PAGE I INTRODUCTION 1 II METHODS AND MATERIALS 1. Preparation of Crude Synaptosomal Membranes from Whole Brain 15 2. Preparation of the To t a l P a r t i c u l a t e F r a c t i o n 15 3. [ 3H] L-Aspartate Binding Assay 18 A. [ 3H] L-Glutamate Binding Assay 21 5. I n h i b i t i o n Curves 21 6. Bio-Rad Protein Assay 23 7. Materials 25 III RESULTS 1. Development of the [ 3H] L-Aspartate Binding Assay a. I n i t i a l experiments 26 b. E f f e c t of cations on the i n i t i a l [ 3H] L-aspartate binding 26 c. Time course of the asso c i a t i o n of [ 3H] L-aspartate binding 29 d. Determination of nondisplaceable [ 3H] L-aspartate binding 29 e. Comparison of [ 3H] L-aspartate binding to synaptosomes and t o t a l p a r t i c u l a t e f r a c t i o n s of whole brain 33 f. E f f e c t of pH and C a 2 + on [ 3H] L-aspartate binding 3A g. E f f e c t of washing the surface of the membrane p e l l e t on displaceable [ 3H] L-aspartate binding 36 2. Characterization of [ 3H] L-Aspartate Binding i n the Cerebellum a. [ 3H] L-aspartate binding i n the cerebellum 39 i . E f f e c t of preincubation on [ 3H] L-aspartate binding to c e r e b e l l a r membranes AO i i . Measurement of [%] L-aspartate binding using a f i l t r a t i o n assay AO b. Time course of the as s o c i a t i o n of [ 3H] L-aspartate binding to c e r e b e l l a r membranes A2 c. Increase of [ 3H] L-aspartate binding with protein concentration A3 d. I n h i b i t i o n of [ 3H] L-aspartate binding to ce r e b e l l a r membranes by L-aspartate A3 e. Scatchard analysis of [ 3H] L-aspartate binding to c e r e b e l l a r membranes A6 3. Pharmacological S p e c i f i c i t y of [%] L-Aspartate and [ 3H] L-Glutamate Binding to Cerebellar Membranes 53 IV DISCUSSION 65 APPENDIX I 83 BIBLIOGRAPHY 8A v i TABLES TABLE I Displaceable [ 3H] L-Aspartate Binding to Crude Synaptosomal Membranes I I E f f e c t of Cations on [ 3H] L-Aspartate Binding III Time Course of Nondisplaceable [ 3H] L-Aspartate Binding IV E f f e c t of pH and C a 2 + on [ 3H] L-Aspartate Binding to Synaptosomal Fractions of Whole Brain V E f f e c t of Washing the Surface of the Membrane P e l l e t on Displaceable [ 3H] L-Aspartate Binding VI E f f e c t of Preincubation on Displaceable [ 3H] L-Aspartate Binding to Cerebellar Membranes VII Nondisplaceable Binding of [ 3H] L-Aspartate to Cerebellar Membranes VIII Summary of Scatchard Analysis Results Shown i n Figures 12 and 13 IX The I C 5 0 Values of Various Compounds Displacing [%] L-Aspartate and [ 3H] L-Glutamate Binding to Cerebellar Membranes X H i l l C o e f f i c i e n t s f o r Various Compounds Displacing [ 3H] L-Aspartate and [ 3H] L-Glutamate Binding to Cerebellar Membranes XI Comparison of the Pharmacological S p e c i f i c i t y of [ 3H] L-Aspartate Binding Obtained by the Author with that Obtained by Sharif and Roberts v i i FIGURES FIGURE P A G E 1 The Three Proposed Points of Attachment of Excitatory Amino Acids 7 Flow Diagram f o r the Preparation of Crude Synaptosomal Membranes 16 3 Flow Diagram f o r the Preparation of To t a l P a r t i c u l a t e 17 Fractions 4 Decrease i n Background Chemiluminescence 20 5 Bio-Rad Protein Standard Curve 24 6 Time Course of [ 3H] L-Aspartate Binding 31 7 [ 3H] L-Aspartate Binding to Crude Synaptosomes and to Total P a r t i c u l a t e Fractions of Whole Brain 32 8 I n h i b i t i o n of [ 3H] L-Aspartate Binding to To t a l P a r t i c u l a t e Fractions of Whole Brain by Unlabelled L-Aspartate 37 9 Time Course of Association of [ 3H] L-Aspartate Binding to Total P a r t i c u l a t e Fractions of Cerebellum 44 10 Increase of [ 3H] L-Aspartate Binding with Protein Concentration 47 11 I n h i b i t i o n of [ 3H] L-Aspartate Binding to To t a l P a r t i c u l a t e Fractions of the Cerebellum by Unlabelled [ 3H] L-Aspartate 48 12 Saturation Analysis of [ 3H] L-Aspartate Binding to To t a l P a r t i c u l a t e Fractions of Cerebellum 50 13 Scatchard Analysis of [ 3H] L-Aspartate Binding to To t a l P a r t i c u l a t e Fractions of Cerebellum 51 14 I n h i b i t i o n of [ 3H] L-Aspartate Binding to Cerebellar Membranes by Various Inhibitors 54-56 15 I n h i b i t i o n of [ 3H] L-Glutamate Binding to Cerebellar Membranes by Various I n h i b i t o r s 57-59 16 H i l l Plot f o r the I n h i b i t i o n of [ 3H] L-Aspartate Binding to Cerebellar Membranes by L-Aspartate 63 17 Possible Drug-Receptor Curves 75 I. INTRODUCTION The n a t u r a l l y occurring amino acid L-aspartate f i r s t became of neuropharmacological i n t e r e s t i n 1960. C u r t i s and coworkers (Curtis et a l . , 1960; Curti s and Koizumi, 1961; C u r t i s and Watkins, 1960; P h i l l i s and Krnjev i c , 1961; Cu r t i s and Davies, 1962) found that i t excited almost a l l neurones i n the mammalian c e n t r a l nervous system. I t was not known i f t h i s e x c i t a t i o n was nonspecific or i f aspartate was a neurotransmitter. Several c r i t e r i a are used to e s t a b l i s h that a substance i s a neurotransmitter (werman, 1966). The c r i t e r i a are: 1. Presence of the substance In neurones, e s p e c i a l l y i n nerve terminals, the s i t e from which neurotransmitters are released. 2. Release of the substance from neurones i n a calcium-dependent magnesium-antagonized manner. Release of a l l substances thought to be neurotransmitters has such i o n i c requirements. 3. S i m i l a r i t y of a c t i o n of the exogenously applied substance with that of the n a t u r a l l y occurring transmitter with respect to a) change i n neuronal f i r i n g rate and i o n i c fluxes associated with changes i n membrane conductance, b) e x c i t a t i o n or i n h i b i t i o n of neurones and c) the e f f e c t s of various pharmacological agents. 4. Presence of an i n a c t i v a t i n g process such as degradation and/or uptake so that the e f f e c t on neurones w i l l terminate r a p i d l y . 5. Presence of a mechanism f o r transmitter synthesis. 6. Demonstration of binding to neuronal or other target membranes since neurotransmitters must bind to receptors located on the membranes to i n i t i a t e t h e i r a c t i o n . 2 None of these c r i t e r i a alone i s s u f f i c i e n t to define a substance as a neurotransmitter. F u l l f i l l m e n t of a l l of these c r i t e r i a , however, provides strong evidence that a substance may be a neurotransmitter. A b r i e f summary follows f o r the evidence that aspartate i s a neurotransmitter. Extensive reviews have been published by Johnson (1978), DeFeudis (1979), N i s t r i and Constanti (1979), P u l l (1981), and Watkins and Evans (1981). Presence Aspartate i s present as a metabolite i n a l l c e l l s . Demonstration of the presence of aspartate i n a neurotransmitter r o l e i s therefore very d i f f i c u l t . Lesion studies i n which c e r t a i n neurones have been depleted, however, provide some evidence to s a t i s f y t h i s c r i t e r i o n . Comparison of the aspartate l e v e l s i n i n t a c t tissues with those i n tissue depleted of neurones i n which there i s evidence f o r a neurotransmitter r o l e for aspartate can be made. If aspartate Is a neurotransmitter i n the neurones i n question, then aspartate l e v e l s w i l l be lower i n lesioned t i s s u e s . This evidence i s much stronger of course i f the only amino acid l e v e l which decreases i s that of aspartate. Only aspartate and glutamate l e v e l s are decreased i n the o l f a c t o r y cortex a f t e r o l f a c t o r y bulb section (Harvey et a l . , 1975). Lesion studies have also demonstrated the presence of aspartate i n the o l i v o c e r e b e l l a r path and dentate nucleus (Perry et a l . , 1977) and i n the do r s a l and v e n t r a l grey matter of the s p i n a l cord (Davidoff et a l . , 1967). Release Neurotransmitter release from neurones can be induced by a p p l i c a t i o n of a s o l u t i o n with a high potassium concentration (usually 50 mM) which induces d e p o l a r i z a t i o n or by e l e c t r i c a l stimulation. Release has been shown with these methods from regions of the c e n t r a l nervous system i n 3 vivo, and i n v i t r o from tis s u e s l i c e s or synaptosomal preparations. Aspartate release has been shown, f o r example, by potassium stimulation from the dendate gyrus (Nadler et a l . , 1977), and by e l e c t r i c a l stimulation from the s p i n a l cord (Roberts and M i t c h e l l , 1972). Release of aspartate has also been shown from the l a t e r a l o l f a c t o r y t r a c t ( C o l l i n s , 1979 a, b), cerebral cortex (Davies and Johnston, 1976), and cerebellum. The aspartate release In the studies above i s dependent on the presence of calcium because of the following i n t e r a c t i o n s . The a c t i o n p o t e n t i a l causes the i n f l u x of calcium i n t o the nerve terminal where the calcium i n t e r a c t s with an actomyosin-like p r o t e i n on the membranes of synaptic v e s i c l e s . These v e s i c l e s then fuse with the plasma membrane and release neurotransmitter by exocytosis. The release i s antagonized by magnesium which competes with calcium f o r passage into the neurone and f o r binding s i t e s once i n s i d e . The magnesium therefore replaces the calcium and prevents the fusion of the synaptic v e s i c l e s with the plasma membrane. In a c t i v a t i o n Aspartate i s i n a c t i v a t e d by uptake into neurones and g l i a (Curtis et a l . , 1970). A low and a high a f f i n i t y uptake system, with -3 -5 average d i s s o c i a t i o n constants of about 2 x 10 M and 2 x 10 M r e s p e c t i v e l y have been demonstrated f o r aspartate (Cox et a l . , 1977; Johnson, 1978). D i s s o c i a t i o n constants are a measure of the a f f i n i t y , that i s the strength of the i n t e r a c t i o n between the receptor and the p a r t i c u l a r compound under examination. Glutamate, an a c i d i c amino acid which d i f f e r s from aspartate by only one carbon atom i n chain length, and which has also been well established as a neurotransmitter, uses the same transport system as aspartate (Balcar and Johnston, 1972; Storm-Mathisen, 1978). A The high a f f i n i t y uptake s i t e s are most l i k e l y those related to neurotransmitter i n a c t i v a t i o n because the aspartate and glutamate concentrations i n rat cerebral s p i n a l f l u i d are 1.98 and 3.6 x 10 ^ M r e s p e c t i v e l y (Clarke and C o l l i n s , 1976). At these concentrations the low a f f i n i t y s i t e i s minimally occupied and therefore does not contribute s i g n i f i c a n t l y to uptake of neurotransmitter. The density of high a f f i n i t y uptake s i t e s a lso c o r r e l a t e s with the density of glutamate binding s i t e s and the regional d i s t r i b u t i o n of i n t r a c e l l u l a r glutamate concentration (Johnson, 1978). The low a f f i n i t y s i t e i s probably related to general metabolic uptake (Levi and R a i t e r i , 1973). The uptake systems are sodium-dependent. Uptake i n the absence of sodium Is n e g l i g i b l e (Davies and Johnston, 1976). Sodium i s transported with aspartate i n t o neurones and g l i a ( S t a l l c u p et a l . , 1979). Synthesis Aspartate Is synthesized i n the t r i c a r b o x y l i c a c i d c y c l e (Benjamin and Quastel, 197A). The two enzymes which are important i n aspartate synthesis are pyruvate carboxylase which converts pyruvate i n t o oxaloacetate and aspartate aminotransferase which converts oxaloacetate to aspartate (Perry et a l . , 1981). These enzymes cannot be used as markers fo r neurones i n which aspartate Is the neurotransmitter because the enzymes are the same as those present i n a l l c e l l s f o r the anabolism of aspartate. Identity of Action Identity of the action of the substance with the n a t u r a l l y occurring transmitter i s one of the more important c r i t e r i a which must be f u l l f i l l e d . I t has been s a t i s f i e d f o r aspartate In many areas of the c e n t r a l nervous system. A p p l i c a t i o n of aspartate by e j e c t i o n from glass microplpettes (iontophoresis) f o r example increases the f i r i n g rate 5 of Renshaw c e l l s i n the s p i n a l cord (Duggan, 1974; Biscoe et a l . , 1976). The e x c i t a t i o n i s consistent with that induced by stimulation of the dorsal roots (Curtis et a l . , 1960). I n t r a c e l l u l a r recordings of aspartate a c t i o n are also consistent with those expected f o r an excitatory neurotransmitter. Aspartate increases membrane conductance by opening sodium channels ( C u r t i s and Johnston, 1974). I n i t i a l l y a l l excitatory amino acids were assumed to act on the same receptor ( C u r t i s and Watkins, 1960; C u r t i s et a l . , 1972). The c a t i o n i c amino group and the two anionic carboxyl groups of a l l excitatory amino acids are thought to Interact i n a three-point attachment to the receptor (Figure 1) (Curtis and Watkins, 1960). It was suggested that more than one of these type of receptors might e x i s t when a d i f f e r e n t i a l s e n s i t i v i t y of neurones to aspartate and glutamate was found (Duggan, 1974). In the cuneate nucleus, f o r example, glutamate potently depolarises sensory terminals while aspartate has no e f f e c t (Davidson and Southwick, 1971). Comparison of the iontophonetic e f f e c t s of aspartate and glutamate themselves on neurones, however, i s not very informative because of the p o s s i b l e c r o s s - r e a c t i v i t y of the two compounds with d i f f e r e n t receptors (Watkins and Evans, 1981). Many compounds have therefore been developed to t r y to d i f f e r e n t i a t e between the actions of the two amino acids i n order to discern whether they i n t e r a c t with the same or separate receptors. The structures of glutamate and aspartate are so s i m i l a r that f i n d i n g agonists and antagonists which are s p e c i f i c to e i t h e r proposed receptor has proven very d i f f i c u l t . Three receptors f o r the e x c i t a t o r y amino acids have now been characterized by t h e i r p r e f e r e n t i a l i n t e r a c t i o n with N-methyl-D-aspartate, quisqualate or kainate ( f o r reviews see: Watkins and Evans, 1981; Watkins 6 e t . a l . , 1981; Watklns, 1981). N-methyl-D-aspartate, a synthetic d e r i v a t i v e of aspartate (Watkins, 1962) reacts very potently and s p e c i f i c a l l y with the N-methyl-D-aspartate active s i t e . Aspartate i s capable of i n t e r a c t i n g with the N-methyl-D-aspartate a c t i v e s i t e . Quisqualate, i s o l a t e d from seeds of a green creeping vine Quisqualis i n d i c a (Takemoto, 1978), Is the most s p e c i f i c glutamate p r e f e r r i n g agonist to date. Kainate i s an analogue of glutamate i n which the conformations of glutamate e x i s t i n the more extended form (Johnston et al.', 1974). The amino-w-carboxylate spacing appears to determine the preference of the d i f f e r e n t compounds f o r the receptor (Figure 1) (McLennan, 1981; McLennan et a l . , 1982). The N-methyl-D-aspartate-preferring receptor seems to accept molecules i n which the spacing i s more extended than those molecules accepted by the glutamate-preferring receptor. Some of the evidence that there are three separate excitatory amino acid receptors i s as follows. I n t r a c e l l u l a r studies with kainate demonstrate that i t has a great potency, slow onset of actio n , produces a very large increase i n membrane conductance, and an i r r e v e r s i b l e d e p o l a r i z a t i o n . These responses are so d i f f e r e n t from those produced by glutamate and aspartate that i t seems u n l i k e l y that kainate Interacts with e i t h e r glutamate or aspartate receptors (Engberg et a l . , 1978). Separate receptors are also indicated by the fa c t that the dose-response curves f o r kainate and glutamate are not p a r a l l e l ( N i s t r i and Constant!, 1979). The strongest evidence that N-methyl-D-aspartate and quisqualate and therefore, as has been proposed, aspartate and glutamate, act at d i f f e r e n t receptors i s that 2-amino-5-phosphonovalerate, the most potent and s p e c i f i c N-methyl-D-aspartate antagonist described to date, s u b s t a n t i a l l y depresses N-methyl-D-aspartate-induced e x c i t a t i o n s while having l i t t l e or no e f f e c t 7 8 on quisqualate-induced e x c i t a t i o n s (Davies et a l . , 1980). Aspartate and glutamate then are mixed agonists i n that both are blocked to some extent by N-methyl-D-aspartate and quisqualate antagonists (Watkins, 1981). Magnesium i s also reported to d i f f e r e n t i a t e between the actions of quisqualate and N-methyl-D-aspartate (Davies et a l . , 1978). The quisqualate responses are not affected by magnesium while N-methyl-D-aspartate responses are i n h i b i t e d by about eighty percent with the same magnesium concentration. Magnesium, however, depresses N-methyl-D-aspartate responses but not aspartate responses possibly because of the proposed i n t e r a c t i o n of aspartate with glutamate p r e f e r r i n g receptors. The hypothesis that separate receptors e x i s t f o r glutamate and aspartate has therefore been l a r g e l y based on the d i f f e r e n t i a l potency of glutamate and aspartate on neurones and the related agonists and antagonists. Iontophoresis, however, i s not necessarily a good technique to use f o r the comparison of potency of various compounds. Differences i n potency may be due to reasons other than the s p e c i f i c i t y of d i f f e r e n t compounds f o r separate receptors such as: 1. the r e l a t i v e numbers of d i f f e r e n t receptor types. I f glutamate and aspartate have the same e f f i c a c y and there are more glutamate than aspartate receptors i n a given area, glutamate might be more e f f e c t i v e than aspartate. 2. d i f f e r e n t e f f i c a c i e s of the compounds at d i f f e r e n t or the same receptors. E f f i c a c y i s a measure of the a b i l i t y of a compound to produce a s p e c i f i e d response. Quisqualate f o r example holds i o n i c channels open longer than glutamate (Anderson et a l . , 1976; Cull-Candy et a l . , 1980) but t h i s i s not strong evidence that quisqualate i s 9 s p e c i f i c f o r glutamate receptors. 3. d i f f e r e n t e f f e c t i v e concentrations of the compounds as a r e s u l t of, for example, d i f f e r e n t rates of i n a c t i v a t i o n of the compounds. Balcar and Johnston (1972) and Cox and coworkers (1977) have pointed out that most amino acid excitants are more potent than glutamate and aspartate because of d i f f e r e n t i a l uptake and not because of d i f f e r e n t e f f i c a c i e s . Another cause of d i f f e r e n t concentrations of compounds i s that the concentration of compound administered can vary up to t h i r t y percent when the same compound i s iontophoresed from d i f f e r e n t b a r r e l s of the same m u l t i b a r r e l l e d electrode ( C u r t i s and Watkins, 1963). Large errors might be encountered when compounds with d i f f e r e n t structures and charges are iontophoresed. The concentration of compound administered by iontophoresis i s therefore not accurately known. 4. d i f f e r e n t s i t e s of a c t i o n of the compounds. One compound, f o r example, may ac t i v a t e s i t e s on the d e n d r i t i c tree so that the response may not be recorded i n the c e l l body whereas another compound may a c t i v a t e s i t e s on the c e l l body where responses are r e a d i l y recorded (BIscoe et a l . , 1976; Davies et a l . , 1978; Usherwood, 1978; Watkins and Evans, 1981; Watkins, 1981). D i f f e r e n t i a t i o n and c h a r a c t e r i z a t i o n of receptors using iontophoretic techniques i s therefore extremely d i f f i c u l t . In 1970, however, the a p p l i c a t i o n of i n v i t r o binding techniques to neurotransmitter receptors began (Snyder, 1978). This technique consists of adding a radioactive neurotransmitter or a r e l a t e d compound to membrane preparations of the region of the c e n t r a l nervous system under i n v e s t i g a t i o n . The t o t a l r a d i o a c t i v i t y bound to such membranes consists of " s p e c i f i c " and "nonspecific" binding components. S p e c i f i c and nonspecific 10 binding are determined by adding a large excess of the unlabelled compound which i s under t e s t to the reaction mixture. The nonspecific material bound i s not displaced by the large excess of added compound and w i l l hereafter more properly be c a l l e d "nondisplaceable binding". Nondisplaceable binding consists of Ionic and nonionic i n t e r a c t i o n s of the compound under t e s t with the membrane fragments. The binding which i s displaced by the unlabelled compound i s the " s p e c i f i c " binding or "displaceable binding". I t consists proposedly of the binding to the neurotransmitter receptor as well as to other s i t e s to which the radioactive compound can be displaced. The neurotransmitter binding s i t e presumably has a higher a f f i n i t y f o r the neurotransmitter s i t e than any other s i t e s from which l a b e l can be displaced. Low concentrations of radioactive neurotransmitter are therefore employed so that neurotransmitter binding s i t e s are p r e f e r e n t i a l l y bound. Concentrations of l a b e l are also kept low to decrease the amount of nondisplaceable binding. Neither the s i t e activated by iontophoresis nor that i n the binding assay are n e c e s s a r i l y the act u a l neurotransmitter receptor. To d i s t i n g u i s h between these s i t e s , those activated by iontophoresis s h a l l be c a l l e d "the active s i t e " , those bound i n the binding assay "the binding s i t e " and those acti v a t e d by the neurotransmitter "the receptor". The binding assay i s a better system than iontophoresis to determine the a f f i n i t y of various compounds f o r the aspartate receptor f o r four major reasons: 1. The concentration of compound applied to the binding s i t e s i s known. A population of binding s i t e s with, f o r example, a high a f f i n i t y can therefore be characterized, i f desired. 2. The concentration and length of time of administration of compound does 11 not change due to i n a c t l v a t l o n by uptake or d i f f u s i o n because: a) the binding assays are performed i n the absence of sodium under which conditions n e g l i g i b l e uptake occurs (Enna and Snyder, 1975) and b) there are no problems with d i f f e r e n t d i f f u s i o n rates i n the binding assay because the compound i s applied d i r e c t l y and uniformly to membrane binding s i t e s suspended i n a homogeneous mixture. The excess compound i s then removed by f i l t r a t i o n or c e n t r i f u g a t i o n a f t e r a predetermined time. 3. The a f f i n i t y of the compound f o r the receptor w i l l be measured whether the binding s i t e occurs on the dendrites or on the c e l l body of neurones. 4. Differences i n e f f i c a c y do not i n t e r f e r e with the binding assay because the binding site-compound i n t e r a c t i o n i s measured and not the response e l i c i t e d a f t e r that i n t e r a c t i o n . 3 To demonstrate that the [ H] L-aspartate i s binding to the s i t e which i s active i n vivo and not just binding n o n s p e c i f i c a l l y , the following c h a r a c t e r i s t i c s must be present (Burt, 1978). The displaceable binding must demonstrate: 1. Saturation; that Is the presence of a f i n i t e number of binding s i t e s . 2. R e v e r s i b i l i t y because the action of aspartate on neurones i n vivo terminates and the receptors are made a v a i l a b l e f o r subsequent a c t i v a t i o n . 3. S t e r e o s p e c i f i c i t y - one isomer binding with much l e s s a f f i n i t y than the other isomer - because many b i o l o g i c a l responses demonstrate s t e r e o s p e c i f i c i t y . 4. lack of a requirement f o r the presence of sodium i n order to d i s t i n g u i s h the neurotransmitter binding s i t e s from uptake s i t e s 12 5. Appropriate regional and s u b c e l l u l a r d i s t r i b u t i o n . Binding to homogenates of brain t i s s u e , f o r example, should be highest i n the synaptic membrane f r a c t i o n s and n e g l i g i b l e i n p u r i f i e d mitochondrial f r a c t i o n s . 6. C o r r e l a t i o n with the actions seen i n vivo; such as a binding a f f i n i t y i n the concentration range where the substance i s active p h y s i o l o g i a l l y and displacement of binding by compounds which demonstrate an i n t e r a c t i o n with the neurotransmitter i n vivo. Displacement of the neurotransmitter by a wide range of concentrations of compound also determines the a f f i n i t y of the compound f o r the neurotransmitter binding s i t e . The a f f i n i t i e s with which a binding s i t e i n t e r a c t s with several compounds give the pharmacological s p e c i f i c i t y . No other binding s i t e w i l l have the same pattern of a f f i n i t i e s . I f the pharmacological s p e c i f i c i t i e s f o r two ligands are d i f f e r e n t , then separate binding s i t e s are indicated, while i f the s p e c i f i c i t i e s are the same, then only one binding s i t e i s indicated. The data from binding studies have demonstrated the existence of separate binding s i t e s f o r kainate and glutamate. The binding studies were 3 performed with [ H] kainate (Simon et a l . , 1976; London and Coyle, 1979) 3 and with [ H] L-glutamate (Roberts, 1974; Foster and Roberts, 1978; Baudry and Lynch, 1979; B i z i e r e et a l . , 1980; Sharif and Roberts, 1980a, b). The pharmacological s p e c i f i c t y of the binding s i t e s f o r the two compounds i n the rat forebrain are d i f f e r e n t (London and Coyle, 1979; B i z i e r e et a l . , 1980). D-glutamate, f o r example, binds to kainate binding s i t e s with one hundred times le s s a f f i n i t y than i t does to glutamate binding s i t e s . Ibotenate and dihydrokainate, however, bind with much greater a f f i n i t y to kainate s i t e s than to glutamate s i t e s . D i f f e r e n t 13 receptors f o r glutamate and kalnate are therefore indicated. The concern of the present study was therefore: 1) to determine the 3 conditions necessary to measure displaceable [ H] L-aspartate binding to membranes prepared from rat brain; 2) to determine whether the binding demonstrated the c h a r a c t e r i s t i c s of binding to the neurotransmitter receptor which i s a c t i v e i n vivo; and 3) a comparison of the 3 pharmacological s p e c i f i c i t y of the [ H] L-aspartate binding with that of 3 [ H] L-glutamate i n order to determine whether the two ligands interacted with separate or i d e n t i c a l s i t e s . During the course of the present work Sharif and Roberts (1981) and 3 Foster and coworkers (1981) reported that [ H] L-aspartate bound displaceably to synaptosomal f r a c t i o n s of the cerebellum and to various s u b c e l l u l a r f r a c t i o n s of the forebrain of the rat r e s p e c t i v e l y . The f r a c t i o n s tested by Foster and coworkers were whole p a r t i c u l a t e , crude mitochondrial (^2^' microsomal, myelin, l i g h t - d e n s i t y synaptic plasma membrane, synaptic plasma membrane and synaptic junction. A neurotransmitter i s expected to bind to the synaptic junction membrane i n order to exert i t s a c t i o n and not to i n t r a c e l l u l a r components or to areas of neurones coated with myelin. In accord with these expectations, Foster and coworkers found that only low l e v e l s of displaceable aspartate binding occurred i n p u r i f i e d f r a c t i o n s of myelin and 3 of mitochondria. The displaceable [ H] L-aspartate binding also increased from whole p a r t i c u l a t e to P2 to synaptic plasma membrane fr a c t i o n s and nine times more binding was seen i n synaptic junction than i n whole p a r t i c u l a t e f r a c t i o n s . The l a t t e r observation Is expected f o r neurotransmitters because i n synaptic junction f r a c t i o n s more binding s i t e s occur per milligram of p r o t e i n than i n whole p a r t i c u l a t e f r a c t i o n s . The 14 whole p a r t i c u l a t e f r a c t i o n s contain a great deal more protein not related to the binding s i t e . 84.5 +9.3 percent of the aspartate binding seen i n synaptic plasma membrane f r a c t i o n s was recovered i n synaptic junction f r a c t i o n s . This i n d i c a t e s that aspartate receptors are concentrated at junctional and not extrajunctional s i t e s . The other r e s u l t s obtained by Sharif and Roberts (1981) and by Foster + 2+ 2+ and coworkers (1981) such as the e f f e c t of K , Ca and Mg on 3 [ H] L-aspartate binding are compared with those of the present study i n the discussion. 15 I I . METHODS AND MATERIALS 1. Preparation of Crude Synaptosomal Membranes from Whole Brain Crude synaptosomal membranes were prepared using a modification of the method of Enna and Snyder (1975). A flow diagram appears i n Figure 2. Male Wistar rats (150 - 350 gm) were k i l l e d by decapitation. The whole brain was removed and homogenized i n 20 volumes (w/v) of i c e - c o l d 0.32 M sucrose with a motor-driven t e f l o n - g l a s s homogenizer (Tri-R homogenizer at 1500 rpm). The membranes were kept i n Ice-cold solutions before use In the binding assay to prevent possible degradation of the binding s i t e s . The homogenate was centrifuged at 1000 x g for 10 minutes (Sorval RC-5 c e n t r i f u g e ) . The r e s u l t i n g p e l l e t was discarded and the supernatant centrifuged at 20000 x g for 20 minutes. The supernatant was discarded and the p e l l e t resuspended i n 20 volumes of cold d i s t i l l e d water by sonication f o r 60 seconds (Sonic Dismembrator s e t t i n g 50 on a scale of 100). This suspension was then centrifuged at 8000 x g f o r 20 minutes, the p e l l e t discarded and the supernatant and buffy coat which i s the upper, l i g h t e r l ayer of the p e l l e t , centrifuged at 48000 x g f o r 20 minutes. The r e s u l t i n g p e l l e t was resuspended In 50 mM Tris-HCl buffer (pH 7.5) by sonication f o r 30 seconds and centrifuged at 48000 x g for 20 minutes. This step was repeated and, unless otherwise stated, the f i n a l p e l l e t was resuspended i n 60 volumes of Tris-HCl buffer. 2. Preparation of the T o t a l P a r t i c u l a t e F r a c t i o n T o t a l p a r t i c u l a t e f r a c t i o n s were prepared using a modification of the methods of Vincent and McGeer (1980) and London and Coyle (1979). Figure 3 shows a flow diagram. Male Wistar r a t s (150 - 350 gm) were k i l l e d by 16 Figure 2 Flow Diagram f o r the Preparation of Crude Synaptosomal Membranes rat brain homogenized i n 0.32 M sucrose 1000 x g 10 min. 20,000 x g 20 min, sonicated 8000 x g 20 min. S, 48000 x g 20 min. sonicated 4 48000 x g 20 5 sonicated 48000 x g 20 min. i P 6 S 6 resuspended i n 60 volumes Tris-HCl 17 Figure 3 Flow Diagram f o r the Preparation of Total P a r t i c u l a t e Fractions rat brain homogenized i n Tris - H C l 48000 x g sonicated X 48000 x g 20 l sonicated 48000 x g 20 min 1 i P„ s„ 3 3 resuspended i n 70 volumes of Tris-HCl 20 min. 18 decapitation. The brain region under i n v e s t i g a t i o n was dissected and homogenized i n 20 volumes of i c e - c o l d 50 mM T r i s - H C l buffer (pH 7.5). This homogenate was centrifuged at 48000 x g f o r 20 minutes. The p e l l e t was resuspended i n buffer by sonication f o r 60 seconds and the suspension centrifuged at 48000 x g f o r 20 minutes. The r e s u l t i n g supernatant was discarded and the p e l l e t resuspended i n buffer by sonication f o r 30 seconds. This suspension was centrifuged at 48000 x g f o r 20 minutes. Unless otherwise stated, the f i n a l p e l l e t was resuspended by sonication f o r 30 seconds i n 70 volumes of Tris-HCl buffer. 3 3. _L H] L-Aspartate Binding Assay 3 Development of the [ H] L-aspartate binding assay i s described i n Section 1 of r e s u l t s . The f i n a l conditions of the binding assay were as follows. The assay mixture was contained i n a t o t a l volume of 1 ml and was composed of 0.5 ml membrane preparation containing approximately 0.35 mg p r o t e i n , 0.4 ml 50 mM Tris-HCl buffer (pH 7.5) or s o l u t i o n of the i n h i b i t o r 3 under t e s t , and 0.1 ml [ H] L-aspartate (98 to 158 nM f i n a l 3 concentration). [ H] L-aspartate and i n h i b i t o r s were prepared i n Tris-HCl and the pH adjusted to 7.5 3 A l l components except the [ H] L-aspartate were added to polypropylene tubes (1.5 ml) stored on i c e . Binding to homogenates was determined i n quadruplicate while that to blank tubes (containing no 3 tissue) was determined i n t r i p l i c a t e . [ H] L-aspartate was then added, the s o l u t i o n mixed and incubated i n a 37° C water bath (Haake E 1) f o r 45 minutes at which time the system had reached equilibrium (see Figure 6). Centrifugation f o r 4 minutes terminated the reaction (Beckman microfuge, Model B). The supernatant was discarded and the p e l l e t rinsed twice with 19 0.1 ml of i c e - c o l d d i s t i l l e d water. Excess water was blotted o f f the p e l l e t and the tubes dri e d with Kimwipes. 1 ml of protosol was added to each tube and l e f t at room temperature u n t i l the ti s s u e was dissolved. The s o l u t i o n was then transferred to a s c i n t i l l a t i o n v i a l and 10 ml of omnifluor (4 gm o m n i f l u o r / l i t r e toluene) added. V i a l s were l e f t at room temperature f o r three hours to allow chemiluminescence produced by mixing protosol and omnifluor to decrease (see Figure 4). The r a d i o a c t i v i t y present was then determined by l i q u i d s c i n t i l l a t i o n spectrometry (Beckman l i q u i d s c i n t i l l a t i o n counter). Counts were corrected f o r e f f i c i e n c y and expressed as d i s i n t e g r a t i o n s per minute (dpm). 3 [ H] L-aspartate binding to tubes was determined by replacing the membrane preparation i n the binding assay with the same volume of Tri s - H C l while background counts were determined by mixing 1 ml of protosol with 10 ml of omnifluor and counting the so l u t i o n a f t e r three hours by l i q u i d s c i n t i l l a t i o n spectrometry. Data were corrected f o r background a c t i v i t y 3 and [ H] L-aspartate binding to tubes. The t o t a l counts added were determined by d i s s o l v i n g 50 y l of the 3 [ H] L-aspartate s o l u t i o n used i n each experiment i n protosol, adding omnifluor and counting on the l i q u i d s c i n t i l l a t i o n counter. A f i n a l 3 concentration of approximately 120 nM [ H] L-aspartate was used because with t h i s concentration displaceable binding was observed and less than 10 percent of the t o t a l r a d i o a c t i v i t y added was bound by homogenates. A v a l i d 3 estimate of the free concentration of [ H] L-aspartate could therefore be made from the t o t a l counts added. Nondisplaceable binding was defined as the binding to membranes i n the -2 presence of 10 M L-asparate. Displaceable binding was determined by subtracting nondisplaceable binding to membranes from the t o t a l binding 20 Figure 4. Decrease of Background Chemiluminescence 10 ml of omnifluor was added to 1 ml of protosol and a c t i v i t y i n the v i a l continuously counted on a Beckman l i q u i d s c i n t i l l a t i o n counter. Counts per minute were measured every 6 minutes, corrected f o r e f f i c i e n c y and expressed as d i s i n t e g r a t i o n s per minute. 2 0 2 2 4 U I 2 0 0 0 I I 0 0 0 I 0 0 0 0 9 0 0 0 8 0 0 0 DPM 7 0 0 0 6 0 0 0 -5 0 0 0 -4 0 0 0 -3 0 0 0 -2 0 0 0 -1 0 0 0 45 9 6 144 192 2 4 0 2 8 8 T I M E ( m i n u t e s ) 21 observed i n the absence of i n h i b i t o r . 3 4. H] L-glutamate Binding Assay 3 The [ H] L-glutamate binding assay was performed exactly as the 3 3 [ H] L-aspartate binding assay except [ H] L-glutamate (18 to 30 nM 3 f i n a l concentration) replaced [ H] L-aspartate and nondisplaceable -2 binding was defined as that i n the presence of 10 M L-glutamate. 45 3 minutes incubation was s u f f i c i e n t l y long f o r the [ H] L-glutamate assay as the reaction had reached equilibrium within 30 minutes (Dr. Andrew Larder, personal communication). 5. I n h i b i t i o n Curves 3 3 I n h i b i t i o n of [ H] L-aspartate (or [ H] L-glutamate) binding by various compounds was determined as follows. Increasing concentrations (0 -2 to 10 M) of the i n h i b i t o r under te s t were incubated with the membrane preparation. The binding to membranes at each concentration was determined and expressed as a percentage of the t o t a l binding i n the absence of i n h i b i t o r . This percentage was then plotted against the log of the i n h i b i t o r concentration. The concentration of i n h i b i t o r at which 50 percent of the displaceable binding was i n h i b i t e d , the IC^n, c o u l d be determined from i n h i b i t i o n curves of each I n h i b i t o r . Comparison of IC^Q values, that i s the a f f i n i t y of each I n h i b i t o r f o r the binding s i t e , could then be made. The l e v e l at which the binding of ligand could not be further decreased by the i n h i b i t o r was taken as the nondisplaceable binding. Drawing the l i n e by hand through the points on the i n h i b i t i o n curves was somewhat a r b i t r a r y . IC,^ values were more reproducibly calculated by 22 conversion of the i n h i b i t i o n curve to a l i n e a r graph by H i l l Transformation. Data obtained from i n h i b i t i o n curves were used to generate the H i l l plot.from the equation 100-y l 0 8^NDB = n l o g 1 " 1 0 8 K ° where y i s the percent of t o t a l binding at each concentration of i n h i b i t o r I, NDB i s the percent of t o t a l binding which i s nondisplaceable, n i s the H i l l c o e f f i c i e n t , and the apparent d i s s o c i a t i o n constant. An example of a H i l l p l o t i s shown In Figure 16. The x-lntercept of the l i n e i s the IC^Q. The slope of the l i n e i s the H i l l c o e f f i c i e n t which i s an i n d i c a t o r of the nature of the i n t e r a c t i o n between the binding s i t e and i n h i b i t o r and i s the main reason f o r generating the H i l l p l o t . When the ligand i n t e r a c t s with a sing l e population of binding s i t e s , the IC^Q curve from 10 +©90 percent of the displaceable binding w i l l f a l l between 2 log u n i t s on the x-axis. A H i l l p l o t with a slope of 1 w i l l be generated. When the ligand i n t e r a c t s with, f o r example, two populations of binding s i t e s , then the IC^Q curve from 10 +©90 percent of the displaceable binding w i l l f a l l between more than 2 log units up to a maximum of 4 log un i t s . The slope of the H i l l p l o t generated i n t h i s instance w i l l be l e s s than one. I f the slope i s 1, therefore, ligand i n t e r a c t i o n with a s i n g l e population of non-interacting s i t e s i s indicated. When the slope i s l e s s than 1, e i t h e r more than one population of s i t e s are involved, each with a d i f f e r e n t a f f i n i t y f o r the ligand, or a sing l e population of s i t e s i s showing negative c o o p e r a t i v i t y where binding of one molecule I n h i b i t s the binding of others. I f the slope i s more than 1, p o s i t i v e cooperativity i n which binding of the i n h i b i t o r to a s i t e enhances subsequent binding i s indic a t e d . The degree to which the H i l l c o e f f i c i e n t i s p o s i t i v e or 23 negative i s not relevant, only that the c o e f f i c i e n t i s p o s i t i v e , negative, or 1. To determine whether each i n h i b i t o r was i n t e r a c t i n g with the same population of s i t e s as those affected by L-aspartate, tubes containing -2 -2 L-aspartate alone (10 M) or L-aspartate (10 M) plus the i n h i b i t o r -2 under te s t (10 M) were assayed with each i n h i b i t i o n curve. If the combination of L-aspartate plus i n h i b i t o r reduced binding more than L-aspartate alone, then some of the i n h i b i t i o n was of the so-called nondisplaceable membrane binding s i t e s . This displacement must then be from a d i f f e r e n t population of s i t e s than that f o r compounds i n h i b i t i n g -2 only the displaceable membrane binding defined by 10 M L-aspartate alone. Comparisons of i n h i b i t o r a f f i n i t y could not accurately be made i n those circumstances. None of the compounds tested i n the present 3 experiments, however, displaced more [ H] L-aspartate than did unlabelled L-aspartate. Binding to the same population of receptors was therefore indicated so that comparison of a l l the compounds tested could be made. 6. Bio-Rad Pr o t e i n Assay Samples of homogenate from each experiment were stored frozen u n t i l assayed f o r the p r o t e i n concentration by the Bio-Rad microassay (Bio-Rad Laboratories, 1979). The t o t a l volume of the assay mixture was 1 ml and consisted of 0.8 ml of d i l u t e d homogenate or standard protein s o l u t i o n , and 0.2 ml of concentrated Bio-Rad dye reagent. Blank tubes used to zero the spectrophotometer contained 0.8 ml of d i s t i l l e d water and 0.2 ml of concentrated dye reagent. 24 Figure 5. Bio-Rad Protein Standard Curve Points are means + standard e r r o r of the mean (SEM) from 10 experiments. 25 Di l u t i o n s of a 100 ug/ml standard s o l u t i o n (Bovine plasma gamma globulin) were made with d i s t i l l e d water to generate a standard curve ranging from 3 to 21 vg/ml protein. Homogenates from experiments were d i l u t e d with d i s t i l l e d water so that the pro t e i n concentration f e l l on the standard curve. When d i l u t i o n s of a l l samples and standards had been made i n duplicate, concentrated dye reagent was added and the so l u t i o n immediately mixed. A f t e r 5 minutes incubation at room temperature, the absorbance of the samples at 595 nm was measured (SP6-500 uv spectrophotometer). Figure 5 shows the standard curve obtained from the average of 10 experiments. Protein concentrations of the samples were determined from the standard curve and expressed i n fmol/mg protein. 7. Materials 3 L-[2, 3- H] a s p a r t i c acid (5.0 to 20 Ci/mmol) was purchased from 3 Amersham, New England Nuclear, or ICN. L-[G- H] glutamic acid was obtained from Amersham, and protosol and omnifluor from New England Nuclear. The following chemicals were also used: L-aspartic acid (K and K Laboratories); D-aspartic acid (Calbiochem); D-a-aminoadipic a c i d , N-methyl-DL-aspartic a c i d , k a i n i c a c i d , and Trisma base (Sigma Chemical Co.); and Standard I-Bovine plasma gamma g l o b u l i n and Bio-Rad dye reagent concentrate (Bio-Rad Laboratories). A l l other reagents were from Fisher S c i e n t i f i c Co. 26 I I I . RESULTS 3 1. Development of the [ H] L-Aspartate Binding Assay l a . I n i t i a l experiments 3 The I n i t i a l [ H] L-aspartate binding assays were performed on synaptosomal preparations of whole b r a i n . The procedure was as described i n the methods except that the incubation time was 30 minutes and the -2 d i s p l a c i n g compound L-aspartate (10 M) was dissolved i n Tris-HCl (pH 7.5) with no further pH adjustment. The average displaceable binding observed (Table I) was 3229 dpm per 0.106 mg p r o t e i n , 0.106 mg being the average amount of p r o t e i n per tube. (See Appendix I f o r a discussion of the low dpm obtained). 3 l b . E f f e c t of cations on the i n i t i a l [ H] L-aspartate binding The low displaceable binding observed i n the i n i t i a l experiments was not considered adequate f o r a r e l i a b l e assay. In an attempt to increase the displaceable binding, a buffer composed of 50 mM Tris-HCl (pH 7.5), 5 mM KC1, 2 mM C a C l 2 , and 1 mM MgCl 2 was tested i n the assay. The displaceable binding was increased 3.6 times i n the buffer containing these s a l t s (Table I I ) . To i d e n t i f y which ions were responsible f o r the increased displaceable binding, the e f f e c t of each ion alone was compared to that In the absence of ions or i n the presence of a l l three ions. The r e s u l t s i n Table I I 2+ l n d i c a t e that Ca appeared mainly responsible f o r the increased 2+ displaceable binding. The increased binding i n the presence of Ca , 3 however, occurred only under the i n i t i a l buffering conditions of the [ H] 27 Table I Displaceable [ H] L-Aspartate Binding to Crude Synaptosomal Membranes Crude synaptosomal membranes suspended i n 50 mM Tris-HCl (pH 7.5) were incubated with [ 3H] L-aspartate (120 nM f i n a l concentration) i n the presence or absence of 10" 2 M L-aspartate, and the displaceable binding determined. Each value i s the mean + SEM of a sin g l e experiment performed i n quadruplicate. Experiment Displaceable [ JH] L-Aspartate Binding (dpm/average mg protein concentration i n the tube) 1 3296 + 397 / 0.0715 mg pr o t e i n 2 4002 + 506 / 0.092 mg pro t e i n 3 2389 + 266 / 0.15 mg protein Mean 3229 + 467 / 0.106 mg protein 28 3 Table II E f f e c t of Cations of [ H] L-Aspartate Binding o Displaceable [ JH] L-aspartate binding was determined i n the presence of 50 mM Tris-HCl buffer (pH 7.5) or the same buffer containing 5 mM KC1, 2 mM CaCl2» and/or 1 mM MgCl2 as s p e c i f i e d below. Values are the mean + SEM from 3 experiments performed i n quadruplicate. S = s i g n i f i c a n t l y d i f f e r e n t from binding i n the absence of s a l t s , p<0.05; NS = not s i g n i f i c a n t l y d i f f e r e n t . Experiment #: Displaceable Binding (fmol/mg protein) 1 2 3 S a l t s i n Buffer None KC1 + MgCl 2 + C a C l 2 KC1 MgCl 2 CaCl„ 689 + 69 1256 + 152 S 232 + 23 NS 673 + 42 NS 1380 + 110 S 342 + 116 1398 + 117 S 268 + 65 NS 309 + 64 NS 588 + 44 NS 476 + 58 2308 + 186 S 389 + 24 NS 553 + 74 NS 1718 + 146 S 29 L-aspartate assay (see Results section I f f o r data and d i s c u s s i o n ) . The + 2+ displaceable binding observed with K or Mg alone was not s i g n i f i c a n t l y d i f f e r e n t from that observed with T r i s buffer (Students t - t e s t ) . Experiments i n sections l c to If of Results were therefore performed with T r i s - H C l plus 2mM C a C l 2 b u f f e r (pH 7.5). Experiments a f t e r section I f , however, were performed using Tris-HCl f o r the reasons o u t l i n e d i n that s e c t i o n . 3 l c Time course of the a s s o c i a t i o n of [ H] L-aspartate binding Analysis of the data from binding assays assumes that the reaction i s at equilibrium when incubation i s terminated. To determine whether the displaceable aspartate binding to synaptosomes of whole brain was at equilibrium a f t e r the 30 minute Incubation i n i t i a l l y chosen, binding was measured a f t e r 0, 5, 10, 15, 20, 30, and 45 minutes incubation. The average binding to tubes (56 dpm) determined from previous experiments was n e g l i g i b l e and therefore not determined i n subsequent experiments (except i n section If of Results). The nondisplaceable binding was maximal i n 5 minutes and d i d not change with longer incubation times (Table I I I ) . Figure 6 shows that displaceable binding was at equilibrium by 30 minutes Incubation. Lower 3 concentrations of [ H] L-aspartate, however, require a longer incubation 3 time. To ensure that equilibrium was reached f o r a l l [ H] L-aspartate concentrations used, a 45 minute incubation time was chosen. 3 Id Determination of nondisplaceable [ H] L-aspartate binding 3 The nondisplaceable binding of [ H] L-aspartate to synaptosomes of 3 whole brain was determined from an i n h i b i t i o n curve with [ H] L-aspartate Table I II Time Course of Nondisplaceable [ ] L-Aspartate Binding Nondisplaceable [ JH] L-aspartate binding (142 nM) to crude synaptosomes of whole brain was determined i n the presence of 10"^ M L-aspartate at various incubation times. Values are the mean + SEM of one experiment performed i n quadruplicate. NS = not s i g n i f i c a n t l y d i f f e r e n t from the nondisplaceable binding at 5 minutes; s = s i g n i f i c a n t l y d i f f e r e n t p<0.05. Incubation Time Nondisplaceable (minutes) Binding (dpm) 5 8216 + 202 10 8173 + 359 NS 15 10096 + 686 S 20 8558 + 139 NS 30 8514 + 276 NS 45 8511 + 212 NS 31 Figure 6. Time Course of [-*H] L-Aspartate Binding Binding to synaptosomal f r a c t i o n s of whole brain incubated f o r various times was determined, o, •, and A represent the r e s u l t s from 3 d i f f e r e n t experiments each performed i n quadruplicate. The SEM of each point ranged from 4% to 63% with the majority below 10%. 5000r 32 Figure 7. [ 3H] L-Aspartate Binding to Crude Synaptosomes and to Total P a r t i c u l a t e Fractions of Whole Brain Membrane preparations were suspended i n Tris-HCl plus 2 mM CaCl2 (pH 7.5). Points are the mean + SEM of 4 experiments. Crude synaptosomal preparations (•), [ 3H] L-aspartate = 146 nM f i n a l concentration. Total p a r t i c u l a t e f r a c t i o n s (o), [ 3H] L-aspartate = 148 nM f i n a l concentration. DISPLACEABLE 8 0 70 BINDING AS P E R C E N T OF T O T A L BINDING 60 50 40 30 20 10 -7 • • • * 6 -5 -4 - 3 -2 L O G [ L - A S P A R T A T E ] 3 3 as the i n h i b i t o r . Figure 7 (closed c i r c l e s ) shows that increasing 3 concentrations of L-aspartate decreased [ H] L-aspartate binding up to - 4 3 x 1 0 M L-aspartate a f t e r which the binding was not further displaced. Nondisplaceable binding comprised 58 percent of the t o t a l -2 binding while the displaceable binding at 1 0 M L-aspartate was 4 3 7 0 fmol per mg p r o t e i n . The IC^Q obtained from H i l l p l o t a n a l y s i s of Figure 7 was 1 1 . 0 uM. The H i l l c o e f f i c i e n t was 0 . 7 9 . 3 l e . Comparison of [ H] L-aspartate binding to synaptosomes and t o t a l p a r t i c u l a t e f r a c t i o n s of whole brain Only 2 5 percent of the i n i t i a l t i s s u e wet weight was recovered a f t e r preparation of crude synaptosomal membranes compared to the 9 0 percent recovery obtained with the t o t a l p a r t i c u l a t e f r a c t i o n . I t was therefore desirable to use t o t a l p a r t i c u l a t e f r a c t i o n s because of the better recovery. Before t o t a l p a r t i c u l a t e f r a c t i o n s could be used, however, i t 3 had to be shown that the [ H] L-aspartate binding s i t e s were the same i n both synaptosomes and t o t a l p a r t i c u l a t e f r a c t i o n s . The i n h i b i t i o n curve i n t o t a l p a r t i c u l a t e f r a c t i o n s of whole b r a i n (Figure 7 , open c i r c l e s ) showed s i m i l a r nondisplaceable binding ( 6 0 percent), IC^ Q ( 5 . 2 0 uM), and H i l l c o e f f i c i e n t ( 0 . 6 0 ) as that observed using crude synaptosomal membranes. IC^Q values d i f f e r i n g by l e s s than a f a c t o r of 1 0 were considered to be s i m i l a r . Differences may have been due to differences i n the membrane f r a c t i o n used. The displaceable binding was increased 2 . 5 times from 1 7 3 0 to 4 3 7 0 fmol/mg protein i n crude synaptosomal preparations as compared to t o t a l p a r t i c u l a t e f r a c t i o n s i n d i c a t i n g that the binding s i t e s may be associated with synaptic junctions. 34 2+ 3 I f . E f f e c t of pH and Ca on [ H] L-aspartate binding In the experiments described so f a r , L-aspartate was dissolved i n -2 Tris-HCl buffer already adjusted to pH 7.5. The pH of a 10 M L-aspartate s o l u t i o n prepared i n t h i s way, however, was found to be 3.5. When the aspartate was added to the binding assay reaction mixture, a f i n a l pH of 4.25 was observed. Receptors i n a binding assay where the pH i s acid may not dis p l a y the 2+ c h a r a c t e r i s t i c s seen i n vivo where the pH i s approximtely neutral. Ca i n the previous experiments may then have acted by s t a b i l i z i n g receptors i n 2+ an acid environment. The increased binding i n the presence of Ca may then not be a c h a r a c t e r i s t i c seen i n vivo. To determine whether displaceable binding would be Increased at a more 2+ 2+ neutral pH (pH 7.5) i n the absence of Ca and whether Ca increased the displaceable binding further at pH 7.5 the following experiment was performed. Four experimental conditions of nondisplaceable binding to -2 synaptosomal membranes were compared. 10 M L-aspartate with the pH of the f i n a l r e a c t i o n mixture e i t h e r 4.2 or 7.5 was dissolved i n e i t h e r Tris-HCl or Tris-HCl plus 2 mM C a C l 2 . The r e s u l t s are shown i n Table IV. Synaptosomal preparations were used again i n the experiments of t h i s section, so that comparisons could be made with previous data. Homogenates incubated with L-aspartate i n T r i s buffer at a f i n a l pH of 2+ 4.2 served as controls. Increasing the pH or adding Ca increased the 2+ displaceable binding but increasing the pH i n the presence of Ca had no 2+ ad d i t i o n a l e f f e c t on displaceable binding. Ca may therefore have acted to s t a b i l i z e receptors under conditions which were too a c i d . It was preferable to use solutions buffered with Tris-HCl ( f i n a l pH 2+ 2+ 7.5) i n the absence of Ca because of the p o s s i b i l i t y that Ca might 35 Table IV E f f e c t of pH and Cal+ on [ JH] L-Aspartate Binding to  Synaptosomal Fractions of Whole Brain Values are the means + SEM of 3 experiments except * which are the r e s u l t s from 2 experiments each performed i n quadruplicate. [%] L-aspartate = 152 nM f i n a l concentration; NDB = nondisplaceable binding; DB = displaceable binding. S = s i g n i f i c a n t l y d i f f e r e n t from binding at pH 4.2 (p<0.05); NS = not s i g n i f i c a n t l y d i f f e r e n t . Buffer Displaceable Binding (fmol/mg protein) Tris-HCl f i n a l pH 4.2 585 + 65, NDB>DB* pH 7.5 1155 + 207 S Tris-HCl + 2 mM C a C l 2 f i n a l pH 4.2 1237 + 366 pH 7.5 1237 + 187 NS 36 substitute f o r Na and thus a c t i v a t e Na -dependent aspartate uptake s i t e s . The i n h i b i t i o n curve generated with L-aspartate solutions adjusted to pH 7 . 5 using t o t a l p a r t i c u l a t e f r a c t i o n s of whole brain i s shown i n Figure 8. The curve with 62 percent nondisplaceable binding, an I C 5 0 of 2.12 pM and H i l l c o e f f i c i e n t of 0.38 was s i m i l a r to the curve generated 2+ i n the presence of Ca at a f i n a l r eaction mixture pH of 4.2 (Figure 7 open c i r c l e s ) . A l l subsequent experiments used t o t a l p a r t i c u l a t e f r a c t i o n s of the br a i n region under i n v e s t i g a t i o n with a l l solutions buffered with 2+ T r i s - H C l , with no Ca present, and the f i n a l pH adjusted to 7 . 5 . l g E f f e c t of washing the surface of the membrane p e l l e t on displaceable 3 _[__ H] L-aspartate binding To determine the e f f e c t of washing the surface of the p e l l e t on the 3 [ H] L-aspartate binding, the p e l l e t was washed 0, 2, or 4 times with 100 u l of i c e - c o l d d i s t i l l e d water and binding i n the presence and -2 absence of 10 M L-aspartate determined (Table V). With no washes, the displaceable binding was very high, as was the standard er ror of the mean. A f t e r 2 washes, displaceable binding was much lower and the SEM was reduced to 7 percent of the displaceable binding. The r e s u l t s obtained a f t e r 4 washes were not s i g n i f i c a n t l y d i f f e r e n t from those with 2 washes. Two washes were therefore performed f o r a l l other experiments. 3 In summary then, the conditions f o r the measurement of [ H] L-aspartate binding u t i l i z e d the t o t a l p a r t i c u l a t e f r a c t i o n s of the brain region under i n v e s t i g a t i o n with a l l solutions made up i n Tris-HCl and the f i n a l r e a c t i o n mixture pH adjusted to 7 . 5 . Two washes of the p e l l e t 37 Figure 8. I n h i b i t i o n of [ JH] L-Aspartate Binding to the Total P a r t i c u l a t e  F r a c t i o n of Whole Brain Membranes were suspended i n Tris-HCl buffer (pH 7.5). [ 3H] L-aspartate = 152 nM. Values are the means + SEM from 3 experiments each performed i n quadruplicate. 38 Table V E f f e c t of Washing the Surface of the Membrane P e l l e t on  Displaceable [%] L-Aspartate Binding The p e l l e t was washed various times with 100 y l of i c e - c o l d d i s t i l l e d H 20 and the displaceable binding of [%] L-aspartate (145 nM f i n a l concentration) to t o t a l p a r t i c u l a t e f r a c t i o n s of whole brain determined. Values are the mean + SEM from a sing l e experiment performed i n quadruplicate. NS = not s i g n i f i c a n t l y d i f f e r e n t from 2 washes, p>0.05. Washes Displaceable Binding (dpm) 0 8593 + 3153 2 5524 + 387 4 4661 + 541 NS 39 obtained a f t e r incubation of the reaction mixture were rou t i n e l y performed. 3 2. Characterization of [ H] L-Aspartate Binding i n the Cerebellum 3 The cerebellum was chosen f o r the c h a r a c t e r i z a t i o n of [ H] L-aspartate binding f o r three major reasons: 1. There i s strong evidence that both aspartate and glutamate are neurotransmitters i n the cerebellum (Storm-Mathisen, 1978; Stone, 1979; Perry et a l . , 1981). Aspartate Is most l i k e l y the neurotransmitter In the climbing f i b e r s (Nadi et a l . , 1977) while glutamate i s most l i k e l y that i n the p a r a l l e l f i b e r s of granule c e l l s . 2. Dissection of the cerebellum i s rapid and very reproducible. Differences between experimental r e s u l t s caused by v a r i a t i o n s i n the brain region dissected are therefore n e g l i g i b l e . 3. The cerebellum i s a large region. Large numbers of rats are therefore not required to do each experiment. 3 2a. J_ H] L-aspartate binding i n the cerebellum 3 The displaceable binding of [ H] L-aspartate to t o t a l p a r t i c u l a t e f r a c t i o n s of cerebellum (730 + 31 fmol/mg protein, n=9) was s i g n i f i c a n t l y greater than i n whole brain preparations (592 + 33 fmol/mg protein, n=5, p<0.05). The average displaceable binding to c e r e b e l l a r membranes was 4468 dpm/0.225 mg protei n , 0.225 mg of p r o t e i n being the average amount of pro t e i n i n each tube. 3 In an attempt to increase the displaceable [ H] L-aspartate binding to c e r e b e l l a r membranes, two procedures were tested: 1. preincubation of 3 the membrane preparation because preincubation increased [ H] L-glutamate binding (Sharif and Roberts, 1980b) and 2. a f i l t r a t i o n assay. 3 2a i . E f f e c t of preincubation on [ H] L-aspartate binding to c e r e b e l l a r  membranes Preincubation was performed during the preparation of the t o t a l p a r t i c u l a t e f r a c t i o n as follows. The cerebellum was homogenized i n i c e - c o l d T r i s - H C l (pH 7.5) and centrifuged at 48000 x g f o r 20 minutes. The p e l l e t , resuspended by sonication f o r 60 seconds, was incubated i n a 37° C water bath f o r 30 minutes. The suspension was then centrifuged at 48000 x g f o r 20 minutes and the membrane preparation continued as i n methods (section 2). Preincubation decreased the displaceable binding by approximately 28 percent (Table VI) so t h i s step was not included i n subsequent experiments. Denaturation of the binding s i t e during preincubation may have been the cause of the decreased binding. 3 2a i i . Measurement of [ H] L-aspartate binding using a f i l t r a t i o n assay The f r a c t i o n of t o t a l binding that i s displaceable can sometimes be increased by decreasing the nondisplaceable binding with the use of f i l t r a t i o n Instead of c e n t r i f u g a t i o n as a means of separating bound from unbound l a b e l . The membrane preparation i s spread over the f i l t e r Instead of being compacted i n t o a p e l l e t . Less l a b e l i s then n o n s p e c i f i c a l l y trapped i n the f i l t r a t i o n method. The technique i s also very f a s t so l e s s d i s s o c i a t i o n of the ligand from the binding s i t e may occur than when ce n t r i f u g a t i o n i s used as the method of separation (Bennett, 1978). 3 A f i l t r a t i o n assay of [ H] L-aspartate binding to t o t a l p a r t i c u l a t e f r a c t i o n s of the cerebellum was performed (kindly by Dr. Andrew Larder) as Table VI E f f e c t of Preincubation on Displaceable [%] L-Aspartate Binding to Cerebellar Membranes Values are the mean + SEM from 2 experiments each performed i n quadruplicate. Preincubation s i g n i f i c a n t l y decreased binding i n both experiments (Students t - t e s t , p<0.05). Experiment Displaceable Binding (fmol/mg protein) 1 2 No preincubation Preincubation 712 + 46 520 + 75 681 + 80 486 + 55 42 follows. The rea c t i o n mixture and Incubation temperature and time were i d e n t i c a l to those i n the c e n t r i f u g a t i o n assay (Methods section 3). At the end of incubation, samples were f i l t e r e d under reduced pressure (12 chambered M i l l i p o r e F i l t r a t i o n Manifold) and the g l a s s - f i b r e f i l t e r s (GF/C f i l t e r s ) washed one to three times with 5 ml each time of i c e - c o l d buffer. The trapped r a d i o a c t i v i t y was measured by soaking the f i l t e r i n protosol (1 ml) f o r 2 hours, adding omnifluor (10 ml) and counting by l i q u i d s c i n t i l l a t i o n spectrometry. Binding to the f i l t e r s i n the presence and -2 absence of L-aspartate (10 M) was also assayed by su b s t i t u t i n g the membrane preparation with the same volume of buffer. For comparison, a ce n t r i f u g a t i o n assay was performed simultaneously with the same membrane preparation used i n the f i l t r a t i o n assay. A f t e r subtracting f i l t e r binding, the displaceable binding i n the f i l t r a t i o n assay performed with one wash was no d i f f e r e n t from that i n the ce n t r i f u g a t i o n assay. The standard e r r o r of the mean of the f i l t r a t i o n assay, however, was high. Two or three washes of the f i l t e r reduced the displaceable binding s i g n i f i c a n t l y below that seen i n the c e n t r i f u g a t i o n assay (Students t - t e s t , p<0.05). Centrifugation therefore continued to be the procedure used to separate bound from unbound lig a n d . 3 2b. Time course of the asso c i a t i o n of [ H] L-aspartate binding to c e r e b e l l a r membranes To determine whether displaceable binding to t o t a l p a r t i c u l a t e f r a c t i o n s of cerebellum was at equilibrium a f t e r the 45 minute incubation 3 time, t o t a l and nondisplaceable [ H] L-aspartate binding was measured a f t e r 0, 5, 10, 15, 20, 30, 45 or 60 minutes incubation. Displaceable binding increased as incubation time increased from 0 to 43 20 minutes a f t e r which no further increase occurred (Figure 9). Binding was therefore at equilibrium at the 45 minute Incubation time chosen f o r 3 [ H] L-aspartate binding studies. The nondisplaceable binding was instantaneous and did not change s i g n i f i c a n t l y with time (Table VII). The time course of binding to the t o t a l p a r t i c u l a t e f r a c t i o n of cerebellum was s i m i l a r to that i n synaptosomal f r a c t i o n s of whole brain 2+ performed i n the presence of Ca (see Figure 6). These data i n f e r that the binding i n cerebellum would be the same i n t o t a l p a r t i c u l a t e f r a c t i o n s and synaptosomal preparations. 3 2c. Increase of [ H] L-aspartate binding with p r o t e i n concentration The p r o t e i n concentration of homogenates varied somewhat between experiments because membranes were resuspended i n buffer according to the wet weight of the p e l l e t . D i f f e r e n t amounts of water were trapped i n p e l l e t s a f t e r the l a s t c e n t r i f u g a t i o n step. To compare r e s u l t s expressed as the binding per milligram of p r o t e i n from d i f f e r e n t experiments, the binding to homogenates must Increase l i n e a r l y with the protein * concentration. T o t a l binding, nondisplaceable binding (that observed i n -2 the presence of 10 M L-aspartate) and displaceable binding to the c e r e b e l l a r t o t a l p a r t i c u l a t e f r a c t i o n were therefore determined at various p r o t e i n concentrations. Figure 10 demonstrates that the displaceable binding increased l i n e a r l y over the range of protein concentrations encountered r o u t i n e l y i n experiments (about 0.25 to 0.45 mg protein/ml). 3 2d. I n h i b i t i o n of [ H] L-aspartate binding to c e r e b e l l a r membranes by  L-aspartate 3 An i n h i b i t i o n curve of [ H] L-aspartate displaced by L-aspartate was 44 Figure 9 . Time Course of Association of [ J H 1 L-Aspartate Binding to T o t a l  P a r t i c u l a t e Fractions of Cerebellum Values are the mean + SEM of a single experiment performed i n quadruplicate. [ 3H] L-aspartate = 140 nM f i n a l concentration. 120O-DISPLACEABLE 10 20 30 40 50 60 INCUBATION TIME (minutes) Table VII Nondisplaceable Binding of [ 3H] L-Aspartate to Cerebellar Membranes Values are the mean + SEM of quadruplicates from a sin g l e experiment. A l l values, except that at 5 minutes, were not s i g n i f i c a n t l y d i f f e r e n t from that at 0 minutes, p>0.05. Incubation Time Nondisplaceable (minutes) Binding (dpm) n 0 6840 + 383 4 5 8587 + 455 4 10 7178 + 266 4 15 6806 + 142 4 20 6236 + 351 4 30 7318 + 323 3 45 8528 + 512 3 60 7675 + 184 3 46 performed on the t o t a l p a r t i c u l a t e f r a c t i o n of cerebellum (Figure 11). —8 — 5 Increasing concentrations of L-aspartate from 10 to 3 x 10 M 3 i n c r e a s i n g l y i n h i b i t e d [ H] L-aspartate binding to the membrane preparation while higher concentrations produced no fur t h e r i n h i b i t i o n . -2 The nondisplaceable binding defined as that occurring at 10 M L-aspartate was 64 percent of the t o t a l binding. The IC^Q calculated from the H i l l plot was 1.81 vM while the H i l l c o e f f i c i e n t was 0.60. 3 These data i n d i c a t e that [ H] L-aspartate binding i n the cerebellum and i n whole brain are s i m i l a r . The apparent d i s s o c i a t i o n constant (K^) f o r an i n h i b i t o r can be calculated from the equation K I -1 + i r -*D (1) where Kp i s the d i s s o c i a t i o n constant of the ligand-receptor complex and L i s the ligand concentration. When the I n h i b i t o r i s the same compound as the ligand, K^ . = K^. Equation (1) becomes :50 and rearranging I C _ = + L *D " I C 5 0 - L s u b s t i t u t i n g the IC,JQ and L values obtained from the i n h i b i t i o n curve of 3 [ H] L-aspartate displaced by L-aspartate, the Kp can be c a l c u l a t e d . The KJJ value f o r L-aspartate was approximately equal to the I C ^ value of 1.81 uM. 3 2e. Saturation a n a l y s i s of [ H] L-aspartate binding to c e r e b e l l a r membranes 3 To determine whether [ H] L-aspartate binding was saturable, 47 Figure 10. Increase of [ JH] L-Aspartate Binding with Protein Concentration T o t a l (•), nondisplaceable (o), and displaceable binding ( A ) to the c e r e b e l l a r t o t a l p a r t i c u l a t e f r a c i o n were determined at various p r o t e i n concentrations. Values are the mean + SEM from one experiment performed i n quadruplicate. [ 3H] L-aspartate = 120 nM f i n a l concentration. 48 Figure 11. I n h i b i t i o n of [ JH] L-Aspartate Binding to the To t a l P a r t i c u l a t e F r a c t i o n of Cerebellum by Unlabelled L-Aspartate Membrane preparations were suspended i n Tri s - H C l buffer (pH 7.5) [ 3H] L-aspartate = 143 nM. Values are the means + SEM from 4 experiments each performed i n quadruplicate. 49 c e r e b e l l a r membranes were Incubated with Increasing concentrations of 3 -2 [ H] L-aspartate i n the presence and absence of 10 M L-aspartate. 3 The displaceable binding observed was plotted as a function of the [ H] L-aspartate concentration (Figure 12). The displaceable binding i n two experiments was saturable while the concentration range i n a t h i r d experiment was not s u f f i c i e n t l y wide to show saturation of displaceable binding. The displaceable binding divided by the free concentration of 3 [ H] L-aspartate was plo t t e d as a function of the free concentration of 3 [ H] L-aspartate to form a Scatchard p l o t . The Scatchard equation i s £ _ Bmax - B F K D i Z ) where B i s the amount of ligand bound, F i s the concentration of ligand, Bmax i s the maximum number of binding s i t e s , and i s the apparent d i s s o c i a t i o n constant. can be derived by rearranging equation 2 Bmax - B K D = B F which i s the negative inverse of the slope of the Scatchard p l o t . Bmax i s the x-intercept of the Scatchard p l o t . The major advantage of using Scatchard a n a l y s i s i s that i n a saturation curve the plateau and therefore Bmax are d i f f i c u l t to determine. Scatchard a n a l y s i s of the data from these three experiments (Figure 13 and Table VIII) gave a mean d i s s o c i a t i o n constant (K^) of 1.64 yM f o r 3 the displaceable [ H] L-aspartate binding and a mean maximum number of binding s i t e s (Bmax) of 7711 fmol/mg protein. The apparent d i s s o c i a t i o n constant determined from Scatchard analysis and from the L-aspartate i n h i b i t i o n curve are therefore i d e n t i c a l . 50 Figure 12. Saturation Analysis of [ H] L-Aspartate Binding to T o t a l  P a r t i c u l a t e Fractions of Cerebellum Values are the means of quadruplicates. The SEM of each point was between 5 and 36 percent with the majority le s s than 15 percent. The l i n e Is the average of r e s u l t s from 3 separate experiments (•, o, A ) . 51 Figure 13. Scatchard Analysis of f-^ H] L-Aspartate Binding to Total  P a r t i c u l a t e Fractions of Cerebellum Points are the means of quadruplicates from three separate experiments while the l i n e through the points i s drawn using the average Kj) and Bmax obtained from the three experiments. 9 0 8 0 7 0 BOUND FRE E 60| (f m o l / m g protelriN nM- J 5 0 4 0 3 0 2 0 10 2000 4000 BOUND ( f m o l / m g p r o t e i n ) 6000 8000 52 Table VIII Summary of Scatchard Analysis Results Shown i n Figures 12 and 13 Experiment # Kn(uM) Bmax (fmol/mg protein) 1 0.971 7358 2 1.87 7245 3 2.07 8531 Mean 1.64 + 0.34 7711 + 411 53 3. Pharmacological S p e c i f i c i t y of [ JH] L-Aspartate and [JH1 L-Glutamate Binding to Cerebellar Membranes 3 3 It i s possible that [ H] L-aspartate and [ H] L-glutamate bind to the same population of binding s i t e s . To t e s t t h i s hypothesis and to 3 characterize the [ H] L-aspartate binding, the a b i l i t y of a range of concentrations of each of L- and D-aspartate, L-glutamate, N-methyl-DL-aspartate, kainate, D- and L-alpha-aminoadipate to i n h i b i t 3 [ H] L-aspartate binding to c e r e b e l l a r membranes was compared with t h e i r 3 a b i l i t y to i n h i b i t [ H] L-glutamate binding. I n h i b i t i o n f o r these 3 3 compounds d i s p l a c i n g [ H] L-aspartate or [ H] L-glutamate are shown i n Figures IA and 1 5 respectively. Where possible, IC50 values and H i l l c o e f f i c i e n t s were determined using the H i l l Transformation (Table IX and X). 3 When [ H] L-aspartate was the ligand, L-aspartate and L-glutamate were equally active with IC^ Q values of 1 .81 and 1 .2A pM r e s p e c t i v e l y 3 and were the most potent i n h i b i t o r s of [ H] L-aspartate binding. L-alpha-aminoadipate was s l i g h t l y l e s s potent (IC^ Q = 7 . 1 2 pM) while D-aspartate with an IC^Q of A 6 . 6 pM was 1 0 times l e s s potent. Kainate 3 and N-methyl-DL-aspartate were very weak i n h i b i t o r s with detectable [ H] L-aspartate displacement only at concentrations greater than 1 0 and 3 0 0 PM r e s p e c t i v e l y . D-alpha-aminoadipate did not i n h i b i t binding even at a concentration of 1 0 0 0 0 PM. At no concentration did kainate or 3 N-methyl-DL-aspartate i n h i b i t 50 percent of the displaceable [ H] L-aspartate binding. values therefore could not be determined f o r kainate, N-methyl-DL-aspartate, or D-alpha-aminoadipate. 3 When the ligand was [ H] L-glutamate, L-glutamate, D-alpha-aminoadipate, and L-aspartate with I C s n values of 2 . 2 5 , 2 . 5 1 , and 54 Figure 14. I n h i b i t i o n of [^H] L-Aspartate Binding to Cerebellar Membranes  by Various I n h i b i t o r s Values are the average of quadruplicates from 2, 3, or 4 experiments as indicated i n Table X. The SEM was between 0 and 11 percent with the majority below 5 percent. The f i n a l concentration of [%] L-aspartate va r i e d between 102 and 153 nM. The squares represent the % of t o t a l binding i n the presence of 10~ 2 M L-aspartate while the c i r c l e s with l i n e s through them represent the % of t o t a l binding i n the presence of L-aspartate (10~ 2 M) plus i n h i b i t o r (10~ 2 M) f o r a) L-glutamate (•, •) and kainate (Q, o), b) D-aspartate (•, •) and N-methyl-DL-aspartate (Q, o), and c) D-alpha-aminoadipate (•, «)and L-alpha-aminoadipate (Q, o). a) BINDING 50 40 30 20-10 • L-glutamate o kainate —» » » 1 i i « « * « • i -3 -2 -8 - 7 -6 -5 -4 LOG [INHIBITOR] 55 14 b) % OF TOTAL BINDING l 0 °* 90 80 70 60 50 40 30 20 10 -9— -©-• D-aspartate o N-methyl-DL-aspartate -8 -7 -6 -5 -4 LOG [INHIBITOR] -3 -2 56 14 c) 1 1 i 1 1 1 > i i i i i_ - 8 -7 -6 -5 -4 -3 -2 L O G [INHIBITOR" 57 Figure 15. I n h i b i t i o n of [ 3H] L-Glutamate Binding to Cerebellar Membranes  by Various I n h i b i t o r s Values are the average of quadruplicates from 2, 3, or 4 experiments as indicated i n Table X. The SEM was between 1 and 12 percent with the majority below 5 percent. The f i n a l concentration of [%] L-glutamate varied between 18 and 30%. The squares represent the % of t o t a l binding i n the presence of 10" 2 M L-glutamate while the c i r c l e s with l i n e s through them represent the % of t o t a l binding i n the presence of L-glutamate (10~ 2 M) plus i n h i b i t o r (10~ 2 M) f o r a) D-aspartate (•, •) and L-aspartate o ), b) kainate (•, •) and L-alpha-aminoadipate (•, o), and c) D-alpha-aminoadipate (•, •) and N-methyl-DL-aspartate (•, o ) . a) 40 30 20 10 • L-glutamate o D-aspartate A L-aspartate -8 -7 -6 -5 -4 LOG [INHIBITOR) -3 -2 15 b) -6 "5 " 4 LOG [INHIBITOR^) 60 Table IX The IC^n Values of Various Compounds Displacing [ 3H] L-Aspartate and [ 3H] L-Glutamate Binding to Cerebellar Membranes IC 5Q values were ca l c u l a t e d by l i n e a r regression analysis of the H i l l p l o t and were averages from 2, 3, or 4 experiments as shown i n Table X. I n h i b i t o r I C 5 0 (PM) [ H] L-Aspartate [ H] L-Glutamate L-aspartate 1.81 3.84 D-aspartate 46.6 36.8 L-glutamate 1.24 2.25 NMDLA >1000 >1000 Kainate >1000 >1000 D-alpha-aminoadipate >10,000 2.51 L-alpha-aminoadipate 7.12 5.28 61 Table X H i l l C o e f f i c i e n t s f o r Various Compounds Displacing [ 3H] L-Aspartate and [ 3H] L-Glutamate Binding to Cerebellar Membranes Values are averages from 2, 3, or 4 experiments (x). Dashes represent compounds f o r which there was i n s u f f i c i e n t i n h i b i t i o n of ligand f o r I C 5 0 value determinations. H i l l C o e f f i c i e n t s I n h i b i t o r 3 [ H] L-Aspartate X 3 [ H] L-Glutamate n L-aspartate 0.60 4 0.60 3 D-aspartate 0.70 2 0.53 3 L-glutamate 0.29 3 0.69 3 NMDLA - 2 - 3 Kainate - 2 - 2 D-alpha-aminoadipate - 3 0.59 3 L-alpha-aminoadipate 0.23 3 0.44 3 3 3 . 8 4 PM resp e c t i v e l y were the most potent i n h i b i t o r s of [ H] L-glutamate binding while D-aspartate with an IC^Q value of 3 6 . 8 PM was an order of magnitude l e s s potent. Kainate and N-methyl-DL-aspartate were 3 very weak i n h i b i t o r s d i s p l a c i n g [ H] L-glutamate binding only at concentrations greater than 1 0 0 and 1 0 0 0 pM res p e c t i v e l y . The H i l l p l o t f o r the L-aspartate i n h i b i t i o n curve i s shown i n Figure 3 1 6 . A l l the H i l l c o e f f i c i e n t s f o r the i n h i b i t i o n of [ H] L-aspartate and 3 [ H] L-glutamate binding to c e r e b e l l a r membranes were l e s s than one (Table X). Negative cooperativity or occupancy of more than one population of s i t e s each with a d i f f e r e n t a f f i n i t y f o r the ligand was therefore indicated. In order to compare data where d i f f e r e n t ligand concentrations are used, I C^Q values are generally converted to d i s s o c i a t i o n constants f o r each i n h i b i t o r by equation ( 1 ) IC. 5 0 ( i ) IC^Q values i n the present experiments, however, could not be converted to true Kj. values as the conversion assumes that one population of non-interacting s i t e s i s bound. The ligand concentration used i n the i n h i b i t i o n experiments, however, was low ( 1 2 0 nM) compared to the TL^ obtained from Scatchard a n a l y s i s . The IC^ Q values f o r the various compounds therefore approximate the values. L- and D-aspartate, L-glutamate and L-alpha-aminoadipate then 3 3 i n h i b i t e d both [ H] L-aspartate and [ H] L-glutamate binding with s i m i l a r potencies. N-methyl-DL-aspartate and kainate did not In h i b i t 3 3 e i t h e r [ H] L-aspartate or [ H] L-glutamate binding. 63 Figure 16. H i l l Plot f o r the I n h i b i t i o n of [ JH] L-aspartate Binding to  Cerebellar Membranes by L-Aspartate Values are obtained from the average of 4 experiments. NDB = Nondisplaceable Binding; y = the percent of t o t a l binding at each concentration of L-aspartate. D-alpha-aminoadipate, however, i n h i b i t e d [ JH] L-glutamate binding much 3 more e f f e c t i v e l y than [ H] L-aspartate. The r e s u l t s from the l i m i t e d number of compounds used to i d e n t i f y the 3 3 pharmacological s p e c i f i c i t y of [ H] L-aspartate and f H] L-glutamate binding indicate the i n t e r a c t i o n of each ligand with d i f f e r e n t s i t e s . Testing more compounds would help to strengthen t h i s conclusion. IV. DISCUSSION The displaceable [""H] L-aspartate binding to t o t a l p a r t i c u l a t e f r a c t i o n s of the cerebellum i n the present experiments demonstrates many of the c h a r a c t e r i s t i c s of a p h y s i o l o g i c a l receptor. The binding i s r e v e r s i b l e , saturable, independent of the presence of Na +, has an a f f i n i t y i n the range where according to iontophoretic studies (Hbsli et a l . , 1973) the neurotransmitter i s a c t i v e i n vivo, demonstrates a pharmacological s p e c i f i c i t y which includes s t e r e o s p e c i f i c i t y , and, as discussed e a r l i e r , has a s u b c e l l u l a r d i s t r i b u t i o n appropriate f o r that of a neurotransmitter binding s i t e . These c h a r a c t e r i s t i c s and the e f f e c t s of 3 ions on the displaceable [ H] L-aspartate binding are discussed i n more d e t a i l below. 3 The displaceable [ H] L-aspartate binding was r e v e r s i b l e In that 3 large q u a n t i t i e s of cold aspartate displaced the bound [ H] L-aspartate. Sh a r i f and Roberts (1981) and Foster and coworkers (1981) also found that 3 [ H] L-aspartate binding to synaptosomal f r a c t i o n s of the cerebellum and to membrane f r a c t i o n s of the forebrain was saturable, r e v e r s i b l e , and independent of Na +. The apparent d i s s o c i a t i o n constants f o r aspartate binding are i n the range of the minimum concentration of aspartate (10 ^ or 10 ^ M) needed 3 to stimulate neurones (Hb'sli et a l . , 1973). Binding of [ H] L-aspartate to synaptic receptors which are a c t i v e i n vivo i s therefore Indicated. 3 The apparent d i s s o c i a t i o n constant (Kp) f o r [ H] L-aspartate obtained by Scatchard analysis i n the present experiments (1.64 + 0.34 uM) Is i n agreement with the 0.874 uM obtained i n crude synaptosomal preparations of cerebellum by Sharif and Roberts (1981). The 66 K D of 0.556 + 0.062 uM obtained by Foster and coworkers (1981) i n p u r i f i e d synaptosomal preparations of the f o r e b r a i n may have been lower than that obtained i n the cerebellum because of the T r i t o n X-100 treatment of the membranes of the forebrain and consequent possible p a r t i a l degradation of the aspartate binding s i t e . - The differ e n c e may also be due 3 to regional v a r i a t i o n i n the a f f i n i t y of [ H] L-aspartate f o r Its binding s i t e . There i s good c o r r e l a t i o n between the apparent d i s s o c i a t i o n constant 3 f o r [ H] L-aspartate binding i n the cerebellum obtained by the author from Scatchard analysis and that calculated from the IC^ Q value obtained from the i n h i b i t i o n curve. The two apparent d i s s o c i a t i o n constants obtained by Sharif and Roberts, however, do not agree. The apparent d i s s o c i a t i o n constant calculated from the IC^Q value (5.0 uM) was 4.68 JiM while that obtained from Scatchard analysis was 0.874 uM. The d i s s o c i a t i o n constant calculated from the IC^Q value obtained by Sharif and Roberts may have displayed a lower a f f i n i t y than that obtained from Scatchard analysis because large concentrations of L-aspartate (up to -2 10 M) were used i n experiments from which the IC^Q values were determined. The Scatchard a n a l y s i s , however, encompassed a lower range of ligand concentrations (from the absence of ligand up to 1000 nM) so that s i t e s with higher a f f i n i t i e s (lower values) were measured. These data 3 i n d i c a t e that at l e a s t two [ H] L-aspartate binding s i t e s e x i s t , one of lower a f f i n i t y than the other. The maximum binding can a l s o be ca l c u l a t e d from the data by Scatchard a n a l y s i s . The maximum binding to t o t a l p a r t i c u l a t e f r a c t i o n s of cerebellum was found i n these experiments to be about s i x times (7.71 pmol/mg) less than that found by Sharif and Roberts i n synaptosomal 67 fr a c t i o n s (44 pmol/mg). More binding i s expected i n synaptosomal preparations because neurotransmitter receptors are concentrated i n t h i s f r a c t i o n . 3 The pharmacological s p e c i f i c i t y of [ H] L-aspartate binding was shown by the IC^Q values obtained f o r the displacement of the binding by 3 several compounds. L-aspartate i n h i b i t e d f H] L-aspartate binding 10 3 times more e f f e c t i v e l y than D-aspartate d i d . The [ H] L-aspartate binding was therefore s t e r e o s p e c i f i c , a c h a r a c t e r i s t i c expected f o r nat u r a l l y occurring reactions. Sharif and Roberts (1981) also found 3 s t e r e o s p e c i f i c i t y of [ H] L-aspartate binding. L-aspartate i n t h e i r experiments was 100 times more e f f e c t i v e than D-aspartate. The IC^ Q values of L-aspartate, L-glutamate, N-methyl-DL-aspartate, and kainate f o r 3 [ H] L-aspartate binding found i n t h i s study were s i m i l a r to those found by Sharif and Roberts (Table X I ) . The I C 5 0 f o r the racemic mixture DL-alpha-aminoadipate obtained by Sharif and Roberts, however, was 100 times l e s s e f f e c t i v e than that obtained by the author f o r e i t h e r isomer alone. The discrepancy between the IC^Q values of D-aspartate, and alpha-aminoadipate obtained i n the present study and those found by Sharif and Roberts may be because, i n the experiments of Sharif and Roberts, the i n h i b i t i o n curves are not well defined. Only four d i f f e r e n t concentrations -5 -3 of from 10 M to 10 M of the various compounds were tested (Roberts et a l . , 1980). As can be seen from Figures 14 and 15 of the present experiments, a great deal of i n h i b i t i o n has already occurred at 10 ^ M of -3 the various compounds under t e s t and 10 M of some compounds, such as 3 D-aspartate, did not f u l l y i n h i b i t the [ H] L-aspartate binding. The i n h i b i t i o n curves derived from such abbreviated 68 Table XI Comparison of the Pharmacological S p e c i f i c i t y of [ 3H] L-Aspartate Binding Obtained by the Author~with that  Obtained by Sharif and Roberts The I C 5 0 values ( i n uM) of the author were taken from Table X but with the use of " i n a c t i v e " defined by Sharif and Roberts (1981) as an I C 5 0 value greater than 10~ 3 M. *Sharif and Roberts used N-methyl-D-aspartate while the author used N-methyl-DL-aspartate. I n h i b i t o r Sharif and Roberts Author L-aspartate D-aspartate L-glutamate N-methylaspartate* Kainate DL-alpha-aminoadipate D-alpha-aminoadipate L-alpha-aminoadipate 5.0 457.0 2.0 i n a c t i v e i n a c t i v e 360.0 1.81 46.6 1.24 in a c t i v e i n a c t i v e i n a c t i v e 7.12 69 data are most l i k e l y s h i f t e d to the r i g h t , producing higher IC^ Q values i n some instances i n the work of Sharif and Roberts (1981) than those obtained i n the present studies. The d i f f e r e n c e s i n production of i n h i b i t i o n curves may be the reason f o r the discrepancy between the H i l l c o e f f i c i e n t s and, consequently, the number of binding s i t e s observed i n the present experiments as compared to those of Sharif and Roberts. In the present experiments more than one L-aspartate binding s i t e i s indicated by the H i l l c o e f f i c i e n t of l e s s than one. The H i l l c o e f f i c i e n t of unity obtained by Sharif and Roberts suggest a homogeneous population of receptors. 3 The H i l l c o e f f i c i e n t s f o r the displacement of [ H] L-aspartate by compounds other than L-aspartate were not determined by Sharif and Roberts. Binding of each of D-aspartate, L-glutamate, and L-alpha-aminoadipate to more than one s i t e , however, i s indicated i n that the H i l l c o e f f i c i e n t s obtained i n the present studies are l e s s than one. The d i f f e r e n t number of binding s i t e s observed by the two inv e s t i g a t i o n s may also have been because t o t a l p a r t i c u l a t e f r a c t i o n s were used i n the present study while synaptosomal f r a c t i o n s were used by Sharif and Roberts. The t o t a l p a r t i c u l a t e f r a c t i o n s may have contained e x t r a -j u n c t i o n a l receptors as w e l l as those found i n synaptosomal f r a c t i o n s . The dif f e r e n c e s i n the IC50 values f o r L-aspartate and alpha-aminoadipate may a l s o have been due to measurement of a lower a f f i n i t y binding s i t e i n the experiments of Sharif and Roberts as discussed e a r l i e r . + 2+ 2+ 3 The e f f e c t s of K , Mg , and Ca on the [ H] L-aspartate binding i n synaptosomal f r a c t i o n s of whole rat b r a i n obtained i n the present study do not agree with those found by Sharif and Roberts (1981) or by Foster and coworkers (1981). Sharif and Roberts found that binding to 70 crude synaptosomal membranes of rat cerebellum was enhanced by K + concentrations of about 2 to 5 mM while concentrations higher than 5 mM i n h i b i t e d the binding. The present study found no e f f e c t of 1 mM K + i n a f i n a l r e a c t i o n mixture of pH 4.5. Sharif and Roberts also found that lower 2+ concentrations of Ca from about 2 to 20 mM enhanced binding while higher concentrations were i n h i b i t i n g . These r e s u l t s f o r the e f f e c t s of 2+ Ca agree with experiments described i n t h i s report i n which the f i n a l 2+ pH was 4.5. At pH 7.5, however, i t was found that Ca had no e f f e c t on 2+ the displaceable aspartate binding. In regard to Mg , Foster and 3 coworkers (1981) found that a concentration of 2.4 mM increased [ H] L-aspartate binding to whole p a r t i c u l a t e f r a c t i o n s of forebrain 2.5 times. 2+ In the present study however, Mg had no e f f e c t on displaceable aspartate binding. These discrepancies could be explained by differences i n the ion concentration, pH, the membrane f r a c t i o n , or the brain region investigated. Differences between membrane subfTactions have been shown by 3 Foster and coworkers (1981). The enhancement of [ H] L-aspartate binding 2+ i n the presence of Ca (2.5 mM) decreased from whole p a r t i c u l a t e f r a c t i o n s of cerebral cortex to p u r i f i e d synaptosomal preparations. C o r r e l a t i o n of the i o n i c e f f e c t s on binding with those seen on de p o l a r i z a t i o n i n vivo are d i f f i c u l t because, i n vivo, the ions a f f e c t s i t e s other than the synaptic receptor. Decreased e x t r a c e l l u l a r K +, f o r example, enhances d e p o l a r i z a t i o n induced by aspartate, a response which i s consistent with the decreased aspartate uptake seen at lower K + concentrations (Evans et a l . , 1977) or the hyperpolarization of neuronal membranes i n low K + concentrations. Assuming from the previous evidence that the s i t e s a c t i v e i n binding aspartate i n v i t r o are those activated i n vivo, the binding assay provides 71 evidence f o r separate aspartate and glutamate receptors. Four pieces of evidence which are discussed i n more d e t a i l below include the d i f f e r e n t pharmacological s p e c i f i c i t i e s , maximum amount of binding, e f f e c t s of ions on the binding, and a l s o the H i l l c o e f f i c i e n t s of les s than one. F i r s t , d i f f e r e n t i a t i o n between L-aspartate and L-glutamate binding i s based p r i m a r i l y on dif f e r e n c e s i n pharmacological s p e c i f i c i t i e s . The apparent d i s s o c i a t i o n constants f o r the various i n h i b i t o r s tested against 3 [ H] L-aspartate binding i n these experiments agreed with those observed i n c e r e b e l l a r synaptosomal f r a c t i o n s by Sharif and Roberts (1981) and i n synaptosomal preparations of the hippocampus by Baudry and Lynch (1979). 3 Diff e r e n t pharmacological s p e c i f i c i t i e s were obtained f o r [ H] 3 L-aspartate and [ H] L-glutamate. The two pharmacological s p e c i f i c i t i e s were found i n the present experiments to d i f f e r i n a f f i n i t y f o r 3 D-alpha-aminoadipate. Sharif and Roberts (1981) found that [ H] 3 L-aspartate and [ H] L-glutamate binding a l s o d i f f e r e d i n the a f f i n i t i e s f o r quisqualate, DL-2-amino-4-phosphonobutyrate, DL-homocysteate, L-glutamate d i e t h y l e s t e r , l-hydroxy-3-aminopyrrolidone-2(HA-966), D-aspartate and somewhat f o r (+)-ibotenate. Second, Sharif and Roberts also found that the maximum binding of glutamate to synaptosomal f r a c t i o n s of the cerebellum was three times higher than that of aspartate to the same membrane preparation. These data ind i c a t e that aspartate and glutamate bind to separate receptors. T h i r d , Foster and coworkers (1981) found that c e r t a i n ions a f f e c t e d aspartate binding d i f f e r e n t l y to glutamate binding. Na + increased 2+ 2+ aspartate binding more than glutamate binding, while Ca and Mg increased glutamate more than aspartate binding. The d i f f e r e n t i a l e f f e c t s of ions on aspartate and glutamate binding lend more evidence to the 72 argument f o r separate receptors. Fourth, the H i l l c o e f f i c i e n t s of l e s s than one obtained i n the present 3 3 study f o r both [ H] L-aspartate and [ H] L-glutamate binding in d i c a t e 3 the i n t e r a c t i o n of each amino acid with more than one s i t e . [ H] L-aspartate may i n t e r a c t with an aspartate' and a glutamate s i t e . How do the data from binding studies c o r r e l a t e with those from iontophoresis? The r e l a t i v e a f f i n i t i e s of L-aspartate, D-aspartate, 3 L-glutamate, and kainate f o r the [ H] L-aspartate binding s i t e are i n accord with the r e l a t i v e potencies of these compounds as seen i n iontophoretic studies. D-aspartate and L-glutamate excite neurones more, l e s s , or as well as L-aspartate depending on the region of the brain tested (Curtis and Watkins, 1960, 1963; H a l l et a l . , 1979). Kainate had l i t t l e 3 e f f e c t on the [ H] L-aspartate binding, which i s consistent with the iontophoretic evidence that kainate acts at a separate receptor (Engberg et a l . , 1978; N i s t r i and Constant!, 1979). There are major disagreements, however, between the iontophoretic and binding data f o r N-methyl-D-aspartate and D- and L-alpha-aminoadipate. Iontophoretic data strongly indicate that aspartate can i n t e r a c t with N-methyl-D-aspartate receptors, although N-methyl-D-aspartate i s much more potent and s p e c i f i c (Evans and Watkins, 1978; Davies and Watkins, 1979) and that D-alpha-aminoadipate has a strong and p r e f e r e n t i a l antagonistic action against N-methyl-D-aspartate (Biscoe et a l . , 1978; McLennan and H a l l , 1978). The D isomer of alpha-aminoadipate i t s e l f i s not e x c i t a t o r y while the L-isomer Is weakly exc i t a t o r y . The L-isomer i s not an antagonist of aspartate or glutamate e x c i t a t i o n (Biscoe et a l . , 1977; Lodge et a l . , 1978). The binding data i n d i c a t e opposite e f f e c t s . N-methyl-D-aspartate and D-alpha-aminoadipate were i n e f f e c t i v e while L-alpha-aminoadipate 73 3 Inhibited the [ H] L-aspartate binding a great deal. Both D- and 3 3 L-alpha-aminoadipate i n h i b i t e d [ H] L-glutamate binding more than [ H] L-aspartate binding. There are several possible reasons f o r the discrepancy between the iontophoretic and binding data. The i n t e r a c t i o n of aspartate with N-methyl-D-aspartate a c t i v e s i t e s determined by iontophoresis may be inc o r r e c t ; a l t e r n a t i v e l y , the binding experiments may not measure the i n t e r a c t i o n with synaptic receptors i n the state and conditions i n which they occur i n vivo. N-methyl-D-aspartate may appear to be more potent than L-aspartate because of d i f f e r e n t i a l uptake of the two compounds. The uptake of L-aspartate (K^ = 1.6 PM) i s much greater than that of N-methyl-D-aspartate (K^ = 28 uM) i n crude synaptosomes of rat hippocampus (Baudry and Lynch, 1979). Af t e r iontophoresis of the same concentrations of L-aspartate and N-methyl-D-aspartate, N-methyl-D-aspartate would therefore be more abundant and would d i f f u s e over a greater area than L-aspartate. N-methyl-D-aspartate would therefore a c t i v a t e more receptors and appear to be more potent than aspartate. N-methyl-D-aspartate, however, may not bind to the receptor as well as L-aspartate, as shown by the r e s u l t s of Garthwaite and Balazs (1981) i n adult rat c e r e b e l l a r s l i c e s . N-methyl-D-aspartate was l e s s e f f e c t i v e than L-aspartate i n increasing cGMP l e v e l s i n c e l l suspensions of rat cerebellum. Only three or four data points, however, were used to determine the dose-response curves from which these conclusions were drawn. Expansion and confirmation of the data i s therefore required. N-methyl-D-aspartate, on the other hand, may not i n t e r a c t with the aspartate receptor but a c t i v a t e a d i f f e r e n t receptor. The cGMP production 74 induced by L-aspartate and L-glutamate was r e s i s t a n t to D-alpha-aminoadipate while N-methyl-D-aspartate-induced stimulation of cGMP was i n h i b i t e d (Garthwaite and Balazs, 1981). I n t r a c e l l u l a r responses are a l s o d i f f e r e n t f o r N-methyl-D-aspartate and L-aspartate. L-aspartate produced a small increase i n membrane conductance while N-methyl-D-aspartate produced a very large decrease (Engberg et a l . , 1978). These data are substantiated by the r e s u l t s of the present 3 experiments i n which N-methyl-D-aspartate had l i t t l e e f f e c t on [ H] L-aspartate binding. Neither the iontophoretic nor the binding studies, however, provide d e f i n i t i v e evidence f o r d i f f e r e n t receptors. The probable a c t i v a t i o n of both glutamate and N-methyl-D-aspartate s i t e s by aspartate, for example, may account f o r the d i f f e r e n t i n t r a c e l l u l a r responses of aspartate and N-methyl-D-aspartate. Another problem with the potencies determined by iontophoresis i s that i n the majority of instances only one e f f e c t i v e dose of, f o r example, aspartate and N-methyl-D-aspartate are compared. The e f f e c t i v e dose i s determined as follows. A range of e j e c t i o n currents f o r aspartate are tested and a current chosen which produces about 50 percent of the maximum response (ED^Q). The potency of N-methyl-D-aspartate as compared to aspartate i s then estimated from the i n t e n s i t y of the iontophoretic current required to e l i c i t an equal response. Only a s i n g l e dose of N-methyl-D-aspartate i s then compared with the ED,.Q of aspartate. In order properly to determine the potencies of various compounds, the ED,JQ values must be compared. The reason f o r the necessity of ED^Q values i s as fol l o w s . Examples of possible dose-response curves f o r two compounds are shown i n Figure s17. At an e j e c t i o n current, 2 (Figure 17a) f o r example, two compounds, A and B, may e l i c i t the same response. At a 75 Figure 17. Possible Dose-Response Curves A and B are two compounds i o n t o p h o r e t i c a l l y ejected at currents 1 and 2. 76 lower e j e c t i o n current, 1, compound A may s t i l l e xhibit the maximum response while compound B e x h i b i t s only 70 percent of the maximum response. The dose-response curves may also be as shown i n Figure 17b where, at e j e c t i o n current 2, compound A i s l e s s potent than compound B but at a much lower e j e c t i o n current, 1, compound B i s l e s s potent than A. Comparison of the potency of various compounds can therefore only be made a f t e r several e j e c t i o n currents of the compounds have been tested, ED^Q curves drawn, and the E D c , n values determined. It i s not possible, however, to obtain ED^Q values i n a large number of instances because i n determining the maximum response the neurone i s inact i v a t e d (Curtis and Watkins, 1960). Relative potencies of various compounds must therefore be viewed with caution. Several problems therefore e x i s t i n the i n t e r p r e t a t i o n of the iontophoretic data. Some of the problems with the binding studies are as follows. In v i t r o studies create an a r t i f i c i a l environment f o r neuronal membranes i n which the aspartate receptors may be a l t e r e d , destroyed, or unable to react properly because of i o n i c or other d e f i c i e n c i e s . The 3 3 [ H] L-aspartate and the [ H] L-glutamate may therefore not have bound to the s i t e s which are activated i n vivo or to s i t e s i n the same state as those i n vivo . The aspartate synaptic receptors may, on the other hand, have been i n t a c t and operable i n the binding assay but because of the low 3 concentration of [ H] L-aspartate employed, only the high a f f i n i t y s i t e s were characterized. Aspartate may i n t e r a c t with lower a f f i n i t y to the 3 N-methyl-D-aspartate binding s i t e so displacement of [ H] L-aspartate by N-methyl-DL-aspartate would not be observed i n the present experiments. In iontophoretic studies low a f f i n i t y s i t e s may have been measured. 77 The disagreement between the data from iontophoretic and binding studies may be because of the measurement of aspartate uptake s i t e s i n the binding studies. Roberts and Kuriyama (1968); Zukin et a l . , (1974); and Enna and Snyder (1975) showed that i n the presence of Na + the neurotransmitter gamma-aminobutyric a c i d (GABA) bound to uptake s i t e s while i n the absence of Na + neurotransmitter s i t e s were ac t i v a t e d . Binding to putative synaptic neurotransmitter receptors i s therefore r o u t i n e l y done i n the absence of Na +. The binding of aspartate to neurotransmitter s i t e s i n the absence of Na +, however, has not been substantiated. + 3 Na -dependent [ H] L-aspartate binding has not been measured, so no c o r r e l a t i o n has been established between Na +-dependent binding and uptake and those d i f f e r e n t i a t e d from Na +-independent binding. Two arguments, however, indi c a t e that i t i s u n l i k e l y that the aspartate binding i n these experiments was to uptake s i t e s . 3 F i r s t , the apparent d i s s o c i a t i o n constants f o r [ H] L-aspartate uptake and Na +-independent binding were d i f f e r e n t . The Kp f o r the high a f f i n i t y [^C]L-aspartate uptake i n r a t c o r t i c a l s l i c e s was 1.6 x 10 M (Davies and Johnston, 1976) while that of Na +-independent aspartate —6 3 binding i n the present study was 10 M. [ H] L-aspartate binding to the uptake s i t e i n the absence of Na + would be l e s s than optimal and the a f f i n i t y therefore l e s s than 10 M. Binding to uptake s i t e s would also have been n e g l i g i b l e i n the present experiments because of the low 3 concentration of [ H] L-aspartate employed (120 nM) compared to the uptake Kp. 3 Second, the pharmacological s p e c i f i c i t y of [ H] L-aspartate and 3 [ H] L-glutamate uptake s i t e s were found by Balcar and Johnston (1972) to 3 be the same. The pharmacological s p e c i f i c i t y of [ H] L-aspartate binding 78 i n the present experiments, however, was d i f f e r e n t from that of glutamate 3 as w e l l as of aspartate uptake s i t e s . In uptake studies [ H] L-aspartate was i n h i b i t e d somewhat more by D-aspartate than by L-aspartate (Balcar and Johnston, 1972). In the present binding studies L-aspartate i n h i b i t s the binding much more than D-aspartate does. There are therefore many possible reasons f o r the discrepancy between the binding and iontophoretic data. C o r r e l a t i o n i s very d i f f i c u l t because the former measures a response which i s s e n s i t i v e to e f f i c a c y and i n a c t i v a t i o n , f o r example, while the l a t t e r measures only the a f f i n i t y of various compounds f o r a binding s i t e which i s unrelated to the c e l l u l a r 3 responses, that i s to e f f i c a c y . The [ H] L-aspartate binding does not demonstrate the c h a r a c t e r i s t i c s of the aspartate actions seen i n vivo such as the strong potency of N-methyl-D-aspartate and therefore may not represent the s i t e which i s activated i n v i v o . The i n vivo studies, however, may not t r u e l y represent the c h a r a c t e r i s t i c s of the aspartate s i t e because of, f o r example, uptake of the compounds iontophoresed. No f i n a l conclusion as to whether separate receptors e x i s t f o r aspartate and glutamate can therefore be made at t h i s time. More data are required from both techniques before a more accurate c o r r e l a t i o n can be made. From iontophoresis, the data required are i n t r a c e l l u l a r recordings, dose-response curves, and more s p e c i f i c agonists and antagonists. The binding studies require c o r r e l a t i o n with a c e l l u l a r response which i s close i n time to the receptor compound i n t e r a c t i o n . Aspartate bound n o n s p e c i f i c a l l y to s i t e s other than the neurotransmitter s i t e would i n t e r f e r e much l e s s with these type of data. The data required from iontophoresis are discussed f i r s t . The s p e c i f i c i t y of most agonists and antagonists, as mentioned 79 e a r l i e r , i s based on e x t r a c e l l u l a r measurements of changes i n f i r i n g rate. Confirmation of the s p e c i f i c i t i e s are required from the more subtle measurements of i n t r a c e l l u l a r recordings such as rev e r s a l p o t e n t i a l s which give information as to which Ionic channels are opened. Differences i n i n t r a c e l l u l a r measurements may d i f f e r e n t i a t e between compounds which were previously not distinguished by the measurement of changes In the f i r i n g rate of neurones. More s p e c i f i c agonists and antagonists f o r aspartate and glutamate are required to d i f f e r e n t i a t e between the actions of the two compounds. Conductance changes produced by aspartate i t s e l f , for example, may be masked i f more glutamate than aspartate receptors are present because of the i n t e r a c t i o n of both amino acids with the two possible receptors. Four types of experiments which would help to r e l a t e the binding studies to an appropriate p h y s i o l o g i c a l receptor are outlined below. F i r s t , c h a r a c t e r i z a t i o n of the N-methyl-D-aspartate binding s i t e may substantiate whether aspartate binds to N-methyl-D-aspartate s i t e s . Unfortunately, Olverman and Watkins (unpublished observations, Watkins and Evans, 1981) and the author could not reproduce the work of Snodgrass 3 (1979) i n which aspartate displaced [ H] N-methyl-D-aspartate binding to membrane f r a c t i o n s of r a t br a i n . Further experiments are therefore necessary f o r better i d e n t i f i c a t i o n of the receptors f o r N-methyl-D-aspartate as w e l l as f o r D-alpha-aminoadipate with aspartate receptors because, as Evans and Watkins (1981) state, "no compelling evidence i s yet a v a i l a b l e to confirm or refute the p o s s i b i l i t y that N-methyl-D-aspartate receptors are aspartate transmitter receptors." A second type of study which could be performed using the binding assay I t s e l f i s one i n which lesions have been made to deplete various 80 neuronal c e l l types. The cerebellum i s an excellent region i n which to study the e f f e c t s of l e s i o n s because animals are a v a i l a b l e i n which c e r t a i n c e r e b e l l a r c e l l types or f i b e r s are absent. Climbing f i b e r s , f o r example, can be depleted with the use of 3-acetylpyridine (Desclin and Escubi, 1974). Third, neurones i n ti s s u e culture may be a good environment i n which to study binding because the receptors would not be subject to possible digestion by enzymes released during membrane preparation. This system, however, may be complicated by the i n a c t i v a t i o n of the compounds under test by uptake. These studies therefore await development of a compound which blocks uptake without blocking the synaptic receptors. The fourth type of experiment i s the d i r e c t measurement of a c e l l u l a r response i n v i t r o such as cGMP l e v e l s and Na + fluxes. Garthwaite and Balazs (1981) measured cGMP l e v e l s In s l i c e s of rat cerebellum i n response to the a p p l i c a t i o n of L-glutamate, L-aspartate, N-methyl-D-aspartate and kainate. They found that the i n v i t r o stimulation of cGMP l e v e l s mimics that seen In vivo. Increases i n cGMP were, f o r example, seen a f t e r i n vivo stimulation of p a r a l l e l or climbing f i b e r s In the cerebellum (Rubin and F e r r e n d e l l i , 1977; Biggio and Gu l d o t t l , 1976; Evans et a l . , 1979). D i f f e r e n t e f f i c a c i e s of various compounds i n a l t e r i n g the cGMP l e v e l s may d i f f e r e n t i a t e between compounds previously not distinguished. Many compounds and regions of the c e n t r a l nervous system have yet to be tested i n the cGMP system. It w i l l be i n t e r e s t i n g to c o r r e l a t e the r e s u l t s with potencies obtained from iontophoretic studies. The measurement of ion fluxes such as Na induced by various compounds i s a technique which looks promising f o r future work. Glutamate, f o r example, changed the Na + fluxes i n s t r i a t a l s l i c e s ( L u i n i et a l . , 1980). Iontophoretic studies suggest that the glutamate receptor, as well 81 as aspartate, are l i n k e d with Na + channels ( C u r t i s and Johnston, 1974). Expansion of these studies with the t e s t i n g of more compounds and the production of dose-response curves would be very i n t e r e s t i n g since the reactions of i o n i c channels are more immediately associated with the receptor-compound i n t e r a c t i o n and therefore more accurately r e f l e c t the i n t e r a c t i o n . Of a l l the experiments mentioned above, those which hold the most promise of c o r r e l a t i o n between data from i n v i t r o and i n vivo experiments are the measurement of dose-response curves f o r Na + fluxes both i n v i t r o i n b r a i n s l i c e s and i n v i v o . 3 The binding assay, assuming that [ H] L-aspartate binds to the receptor which i s a c t i v e i n vivo and that agonists and antagonists s p e c i f i c to the s i t e are found, could provide a great deal of information. The d i s t r i b u t i o n , r e g u lation, development, a l t e r a t i o n during disease or aging, and the screening of various drugs f o r e f f e c t s on the aspartate receptor could be studied. D i s t r i b u t i o n and l e s i o n studies may help to determine the pathways i n which aspartate may be a neurotransmitter. The l o c a l i z a t i o n of aspartate receptors at pre- or post-synaptic, j u n c t i o n a l or extrajunctional s i t e s could be determined by autoradiography or histochemistry. The point during formation of the synapse at which receptors become evident and any changes which may occur during development could a l s o be studied with binding assays, p a r t i c u l a r l y Scatchard analysis of the binding at each stage of development. The possible a l t e r a t i o n of receptors during prolonged administration of c e r t a i n drugs could also be determined from binding studies. These data may be correlated with changes i n p h y s i o l o g i c a l responses such as Na + fluxes or cGMP l e v e l s . Before any of these studies can proceed, however, i d e n t i f i c a t i o n of the aspartate binding s i t e with the receptor which i s active i n vivo i s required. 83 APPENDIX I The d i s i n t e g r a t i o n s per minute obtained f o r [ JH] L-aspartate binding were low because of the low s p e c i f i c a c t i v i t y of the l a b e l used. The free 3 concentration of [ H] L-aspartate could not be estimated when concentrations i n which greater than 10 percent of the t o t a l r a d i o a c t i v i t y bound to membrane preparations were used. Higher concentrations of l a b e l also dramatically increase the nondisplaceable binding so that accurate measurements of the displaceable binding, which i s determined by subtracting the nondisplaceable from the t o t a l binding, would be very d i f f i c u l t . Higher concentrations of t h i s l a b e l could therefore not be used to increase the di s i n t e g r a t i o n s per minute obtained. 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