Gen. Pharmac.• 1976. Vol. 7, pp. 5 to 14. Pergamon Press. Printed in Great Britain

MINIREVIEW RECEPTORS FOR AMINO ACIDS IN EXCITABLE TISSUES N. T. Buu, E. PUlL AND N. M. VAN GELDER Institut de diagnostic et de recherches cliniques et Groupe de recherche en sciences neurologiques du Conseil de recherches medicales, Department de physiologie, Universite de Montreal, Canada (Received 16 June 1975)

Ever since Bazemore et al. (1957) identified GABA as the principal inhibitory substance in brain extracts, a large number of analogues have been studied to ascertain why this particular amino acid has such a strong depressant action on excitable membranes. The extensive surveys by Curtis & Watkins (1960, 1965) have subsequently indicated that relatively few amino acids, applied externally, are able to directly influence neuronal excitability. During the past fiteen years, an enormous amount of literature has accumulated which deals with the physiological pharmacological and biochemical properties of these special amino acids. Two comprehensive reviews have recently appeared on the subject (Curtis & Johnston, 1974; Krnjevic, 1974). This brief review will not attempt to duplicate these accomplishments but will re-examine existing data on the relationship between structural and other physicochemical properties of amino acids and their ability to induce an effect in excitable tissues.

1971; see also Curtis & Watkins, 1960) that each type of receptor must incorporate an additional third active center close to, and at a fixed distance from the amino site. This suggestion was based on the observation that the substituent on the carbon atom adjacent to the amino group (C~ in glutamate, C y in GABA) appeared to be the sole determinant for physiological activity (Fig. 1). Replacement of the carboxyl group by a sterically smaller and chemically inert (not capable of H-bonding) hydrogen atom transforms an excitatory amino acid into one having a depressant action; with diminishing steric influence of that region the physiological effect of the derivative gradually decreases. Close examination of the steric dimensions of that region of the molecule (Fig. 2) has provided the following information: (i) The a-carboxyl group of glutamic acid seems essential for the increase in sodium permeability. The y-hydrogen of GABA is essential for causing an increase in chloride permeability (see Fig. 1). (ii) Hydrogen bonding of the amino and acarboxyl groups of glutamic acid with their respective receptor sites, separates the two sites by a distance of approximately 6·3A. That region of the GABA molecule with the y-hydrogen, when combined with the amino site of its receptor, occupies in the membrane a space of approximately 3·5 A in dia. (iii) The diameters of hydrated sodium and chloride ions have been estimated at 4·8Q-5·12A and 3,32-3,86 A respectively (Lettvin et al., 1964; Guyton, 1968). The length and substitution of the carbon chain (e.g. C B and C y , Fig. 2) between the w-acidic group and the amino group alters the potency of such substances but does not modify their inherent ability to cause either the excitatory or the depressant effect on excitable membranes. In general, replacement of the freely-rotating carbon chain by a rigid ring structure such as ibotenic acid (excitatory) or muscimol (inhibitory), L-a-kainic acid and DL-quisqualic acid, results in substances which are more potent than glutamate or GABA (see Johnston et al., 1968, 1974; Shinozaki & Konishi, 1970; Shinozaki & Shibuya, 1974a). In many compounds of this class, the acidic nature of the ro-acidic group is often enhanced which likely increases the affinity of such

STRUCTURE-ACTIVITY RELATIONSHIPS

All amino acids capable of a direct action on excitable membranes possess a structure incorporating a free amino group and an ionizable acidic group (-COOH; -SOsH; -SOsH) separated by a distance equivalent to a straight carbon chain of one to four carbon atoms (~2-6A). With the exception of glycine, the other amino acids in this class appear to form natural pairs of excitatory and inhibitory substances: L-aspartic acid and l3-alanine, L-cysteic acid and taurine, L-glutamic acid and GABA. The excitatory dicarboxylic amino acid is transformed into the corresponding inhibitory OJ-amino acid by a-decarboxylation. In order for these amino acids to influence membrane excitability, all ionizable groups i.e. the amino group (NH s +), the a-carboxyl (COO-) and the OJ-carboxyl, sulfinic (SOs -) or sulfonic group (SOs -), must remain unsubstituted and ionized at physiological pH. Both the excitatory and inhibitory receptors likely have two active centers in common, namely those binding the amino group and the OJ-acidic group respectively. It has been suggested (van Gelder, 5

6

N. T. Boo, E. Pun. AND N. M.

__~~'~_'T'''-- glutamate

1(",'0 ACTIVITY:++++

•

~

cod

,l

VAN GELDER

interfere with, or may completely prevent the amino acid from reaching the receptor (Fig. 2). This depends on which 13 or y (a) hydrogen has been substituted and whether a conformational change in molecular configuration can compensate for this interference (see below).

c'

C'

H

~,--l ~_

cooT'T T

y- aminobutyrat. ACTIVITy,

_

8,hydrazlnopropionat. Kl'lO'7", ACTIVITY,_

-

amlnoaxypropionote KI-l0-SM

ACTIVITY,NONE

coo~o c'

H,

PROPERTIES OF THE RECEPTOR PROTEIN

1(",'10,3",

aminooxyacetat. KI'lO""'",

ACTIVITY, NONE

Fig. 1. Dreiding models of glutamate and GABA together with three closely related structural analogues. The co-amino acids only differ with respect to the configuration around the first atom adjacent to the amino group. The same region of the molecule appears to determine exclusively the pharmacological action of the amino acids. Insertion of an additional methylenic group between the terminal carboxyl and amino group of glutamate or GABA practically abolishes pharmacological activity. One more carbon renders the compounds inactive. At physiological pH the physicochemical properties of hydrazinopropionate, aminooxypropionate, and aminooxyacetate, are very similar with respect to GABA. Values of K (Km or K,) refer to "apparent affinity constants" of these substances for GABA aminotransferase. The affinity of such compounds for the enzyme is unrelated to the structural requirements for physiological action. analogues for the receptor surface. Thus a prolongation and enhancement of the excitatory effect is observed when the co-carboxyl of L-glutamic acid is replaced by a more acidic, sulfonic group (L-homocysteic acid; cf. Curtis & Watkins, 1963). On the other hand, potency is attenuated when one of the hydrogens in the intervening carbon chain of GABA is substituted by a sterically larger hydroxyl, chloride or phenyl moeity (cf. Bazemore et al., 1957; Buu & van Gelder, 1974; Davies & Watkins, 1974). An increase in chain length beyond four carbon atoms has a similar effect (Curtis & Watkins, 1960). Both types of modifications have in common that the steric volume of the molecule increases. This factor, therefore, must also influence the proper complement of the amino acid to its receptor surface. A large substituent on the molecule may simply

An a-decarboxylation of an excitatory amino acid yields an inhibitory homologue and a single receptor protein should theoretically be capable of binding both classes of amino acids (van Gelder, 1971). Alternatively, separate receptors for these amino acids may exist. De Robertis & Fiszer de Plazas (1974) and Fiszer de Plazas & De Robertis (1 974) reported the isolation of two different proteolipids from shrimp muscle. One of these binds GABA (a process prevented by bicuculline) but not glutamic acid. The other hydrophobic protein only interacts with glutamic acid which binding is prevented by a-methyl-oL-glutamic acid. Bicuculline specifically blocks the GABA receptor (Curtis etal., 1970) whereas a-methyl-oL-glutamic acid appears to hinder access of glutamic acid to its receptor (see position H~, Fig. 2C). Such findings and the studies by Takeuchi & Takeuchi (1965, 1966) indicate that the receptor protein or membrane sites which are sensitive to GABA, are clearly distinguishable from those binding glutamic acid. These investigators demonstrated that the crayfish muscle became desensitized to repeated applications of L-glutamic acid, or to the effects of repetitive stimulation of the excitatory nerve, but at the same time remained sensitive to GABA. In addition, little interaction appeared to occur between GABA and the receptors for L-glutamate, or vice versa. Two distinct populations of membrane receptors (or sites) for L-glutamic acid are suggested by the studies of Usherwood and collaborators (Cull-Candy & Usherwood, 1973; Lea & Usherwood, 1973a, 1973b; Shinozaki & Shibuya, 1974b) in certain types of invertebrate muscle, and by others in molluscan neurones (Szczepaniak & Cottrell, 1973; Kerkut et at., 1975; see also Gerschenfeld & Lasansky, 1964; Oomura et al., 1974). The first type of receptor is found outside the synaptic region of locust muscle; it is sensitive to ibotenic acid (rigidlyextended analogue of glutamate; Lea & Usherwood, 1973a, 1973b). This receptor may be identical to the glutamate-sensitive inhibitory receptor in molluscan neurones which can be blocked by OL-aaminopimelic acid (Kerkut et al., 1975). Activation of this receptor by glutamic acid enhances permeability to chloride (and possibly potassium permeability; Kerkut et al., 1969), resulting either in a hyperpolarizing or depolarizing response of the cell. The effect of course depends on the equilibrium potential for chloride of the cell vis a vis its resting potential (Lea & Usherwood, 1973b; Hironaka, 1974).

A

B

coo"

y -aminobutyrate

glutamate

c

···············T

0

0

1.00 A

Tor···· 1.... . .. ..1

....... .J..

2.65 A

T

o

2.05 A

.

:~.l:· ~::::i'

2.45 0

...

--~

~ ~~~~T

H

:'.'

'Y

~~H'Y ~

........1. .

Fig. 2. Stuart models of glutamate (A) and GABA (B) demonstrating the difference in configuration of the amino acids around the carbon, adjacent to the amino group. The nature of the substituent at Co (i.e. C y of GABA) exclusively determines whether Ul-amino acids exhibit inhibitory or excitatory activity. In C and D, Dreiding models of the Co region of glutamate and the C y region of GABA indicate that bond angle restriction does not allow alteration in the position of COO o or Hy relative to the amino group. Due to the steric dimensions of these rigid configurations, surrounding molecules or membrane structures interacting with glutamate must be separated by a minimum distance of 5·70 A whereas for GABA this same distance will be approximately 4·50 A (Cohn & Edsall, 1943). By postulating hydrogen bonding (2,76 A) between COOo of glutamate with a binding site in the membrane and a I· 2 A bond between the amino group and its site (Pauling, 1960), the separation between the two sites upon binding of glutamate becomes approximately 6·30 A. The distance between an amino site in a GABA receptor and protein structures conforming to the steric influence of H y narrows to 3·65 A. The hydrogen atom (H y ) replacing COO. has been given a different pattern to emphasize its functional significance. Reproduced by permission of the National Research Council of Canada from the Canadian Journal of Physiology and Pharmacology, Volume 49,1971. pp.513-519.

Receptors for amino acids in excitable tissues The other glutamate receptor, present postjunctionally in the synaptic region, probably mediates an increase in sodium conductance (depolarizing) as suggested by the positive reversal potential for the action of L-glutamate (Takeuchi & Onodera, 1973; Anwyl & Usherwood, 1974). In excellent agreement with these findings, Lunt (1973) obtained two different hydrophobic proteins from the locust muscle, both of which demonstrated a high and rather specific affinity for glutamic acid. The existence of more than one distinct glutamate receptor in the vertebrate central nervous system can be suggested only on the basis of less direct evidence. In intracellular investigations, combinations of hyperpolarizing and depolarizing responses have been recorded upon local applications of glutamic acid to neurones in the fish retina (Sugawara & Negishi, 1973; Murakami et al., 1975), and in the cat spinal cord (Bernardi et at., 1972; Zieglgansberger & PuiI, 1973a). Although such biphasic responses are compatible with the data from the invertebrate systems, the unequivocal presence of two different receptors for glutamate in neurones of vertebrates clearly requires more study. In cases where their existence is proven, it seems worthwhile investigating whether an enhanced chloride permeability may be due to rapid decarboxylation of glutamic acid to GABA at the relevant receptor sites (cf. Usherwood & Machili, 1968). The above observations indicate that the changes in membrane properties brought about by the action of L-glutamic acid, primarily affect the chloride or sodium permeability. (There is evidence as well in Onchidium neurones of a glutamate receptor mediating an increase in potassium permeability; Oomura et al., 1974.) Several investigations, however, have indicated that Ca 2+ may also playa role in the resulting conductance change. For example, Ames et al. (1967) have suggested that the specific reaction between glutamic acid and a membrane component involves Ca 2 + and Na + influx. Also, voltage clamp studies of the action of L-glutamate on muscle (Takeuchi & Onodera, 1973; Anwyl & Usherwood, 1974) have demonstrated a small contribution by Ca 2 + to the inward current carried mostly by Na +. It is presently very difficult to determine if L-glutamic acid initiates a permeability change to Na + by displacing Ca 2 + from membrane sites which control sodium permeability (see Tan, 1975). At the same time, the sodium permeability change caused by L-glutamic acid appears to be distinct from the Na + ionophore formation that occurs during generation of the action potential. Tetrodotoxin, a specific inactivator of the Na + ionophores associated with spike generation, does not interfere with the depolarizing actions of Lglutamate (Zieglgansberger and Puil, 1972) or nLhomocysteate (Curtis et al., 1972). Such data provide evidence that tetrodotoxin blocks electrical excitability but not the chemically-excitable Na +

7

channels. The latter appear to be directly dependent on the interaction of glutamate with a receptor.

AMINO ACID-RECEPTOR INTERACTION

Recently, McCulloch et al. (1974) have postulated the existence of one excitatory receptor reacting with the extended form of glutamic acid, thus excluding aspartic acid from this receptor (Fig. 1, glutamic acid vs amino-oxyacetic acid). This type of receptor predominates in spinal interneurones. Another (excitatory) receptor, in Renshaw cells, presumably interacts with N-methyl-n-aspartic acid, L-aspartic acid and L-glutamic acid (folded). Lea & Usherwood (1973a) proposed two active glutamic acid conformations in order to account for the insensitivity of one of the glutamate receptors to ibotenic acid. According to their hypothesis, extrasynaptic, "chloride receptors" would accept glutamic acid in the extended configuration, and ibotenic acid. The synaptic receptor mediating an increase in sodium conductance would direct glutamic acid to assume the folded configuration (Fig. 2 A; van Gelder, 1971). It is useful to re-examine the problem of amino acid-receptor interaction, taking into account the essential difference in configuration between the L- and n-forms of an amino acid. Bearing in mind that the a-carboxyl, a-amino configuration of glutamic acid and its analogues is invariable and fixed (Fig. 2C), all such substances must combine with two complementary active centers of a receptor in the same manner. It then becomes apparent that L-aspartic acid, L-glutamic acid and n-glutamic acid can combine with the same receptor surface (Fig. 3). However, n-aspartic acid, and thus N-methyl-naspartic acid, are less likely to combine with this receptor because of (a) the reversed orientation of the a-carboxyl group (up instead of down) and (b) the steric interference of the H~ hydrogen (down instead of up). In the case of n-glutamic acid, the greater flexibility of the carbon chain partially obviates this interference (Fig. 3, right). Examination of cyclic analogues of glutamic acid reveals other interesting information. A comparison of the L- and n-configurations of ibotenic acid, kainic acid and quisquaiic acid (Fig. 4) affords the following conclusions: (i) Due to its invariable configuration, the receptor for L-ibotenic acid (chloride receptor) is clearly distinct from the "sodium receptor" which binds L-aspartic acid and, hence, folded L-glutamic acid (Fig. 4, teft). The same chloride receptor can also bind the extended conformation of L-quisquaiic acid. (ii) In the folded conformation both L- and n-quisquaiic acid may combine with the sodium receptor (Fig. 4, right). The n-isomer does not appear able to interact with the chloride receptor.

N. T. Boo, E. PulL AND N. M.

8

VAN GELDER

CO

o. Nlll

CH@-

L·ASPARTIC ACID

D·GLUTAMIC ACID

L·GLUTAMIC ACID

D·GLUTAMIC ACID

Fig. 3. Orientation of L-aspartic acid and of D- and L-glutamic acid to a hypothetical receptor surface incorporating three reactive centers. Left: L-aspartic acid and L-glutamic acid (folded) may combine with the same three sites of the receptor. The position of the sites relative to one another is assumed from the maximum distance between the carboxyl groups of L-aspartic acid (invariable because of bond angle restrictions). Right: Extended configuration of D-glutamic acid (thus also D-aspartic acid) cannot combine with the same receptor because of changed orientation of the a-carboxyl group (up) and steric hindrance exerted by Ha (down). The greater flexibility of the carbon chain of D-glutamic acid, in comparison to D-aspartic acid, permits the folded configuration of D-glutamic acid (lower right) to combine with this receptor but H a will still exert some steric interference.

(iii) The maximally extended conformation of L-a-kainic acid (Fig. 4, left) complements neither the chloride nor the sodium receptor (note orientation of y-hydrogens). Somewhat rotated and folded to remove the steric hindrance of the y-hydrogens, L-a-kainic acid may react with the sodium receptor. The n-isomer, as in the case of D-ibotenic acid, does not possess the proper configuration for one or the other type ofreceptor. Shinozaki & Shibuya (1974b) have reported that in crayfish muscle L-a-kainic acid potentiates the action of L-glutamic acid and of L-aspartic acid, but had only a slight depolarizing effect of its own. The action of quisqualic acid was not influenced. (iv) When L-glutamic acid is maximally extended (see Fig. 1) the p-hydrogens appear to preclude complementarity of the y-carboxyl group to the chloride receptor. Theoretically, therefore, it seems unlikely that the three reactive groups of extended L-glutamic acid bind simultaneously to a chloride receptor. The fact that the folded D- and L-isomers of quisqualic acid are equally effective in combining with the same receptor may partly explain why a racemic solution of this analogue is so extremely potent in producing excitation (Shinozaki & Shibuya. 1974a). Racemic solutions of other glutamic acid analogues, in contrast, may in reality contain only 50 per cent of the concentration of the pharmacologically active isomer.

The preferred conformation of GABA for combination with its receptor is still somewhat in dispute. However, investigations by Johnston and his collaborators especially, over the past few years seem to lead to an overall conclusion that this amino acid, and perhaps also taurine, combines with a receptor in an extended configuration. The most convincing evidence is derived from the observation that muscimol has, like GABA, a strong depressant action on neurones (Johnston et al., 1968; Walker et al., 1971), and on insect muscle fibers (Lea & Usherwood, 1973a). This substance is the decarboxylation product of ibotenic acid and therefore also possesses a rigidly extended configuration (Fig. 4; Kier & Truitt, 1970). Johnston et al. (1975) have also compared the action of two isomeric forms of 4-aminocrotonic acid, which is a semi-rigid, ethylenic analogue of GABA. In the cis-conformation this compound represents the folded configuration of GABA whereas the trans-isomer is analogous to the extended form of GABA. Unfortunately, both the cis- and trans-isomers depress the activity of spinal motoneurones. Only the depressant activity of trans-4-aminocrotonic acid is believed to be similar to that of GABA because, unlike the cis-form, its action is blocked by bicuculline. According to Beart et al. (1971), bicuculIine blocks the GABA receptor in its extended conformation. The opposite conclusion was reached by Stewart et al. (1971), namely that both GABA and

Receptors for amino acids in excitable tissues

CO N@

oe

CH~

L-IBOTENIC ACID

'C L-QUISQUALIC ACID

L-a-KAINIC ACID

D-QUISQUALIC ACID

•

L-QUISQUALIC ACID

L- a-KAINIC ACID

Fig. 4. Dreiding models of three analogues of glutamic acid which possess partially rigid configurations. Left: Invariable, extended conformation of L-ibotenic acid probably prevents its interaction with the receptor which accepts L-aspartic acid and folded L-glutamic acid. A different receptor is postulated which also accepts L-quisqualic acid when maximally extended (see text). On the other hand, L-a-kainic acid appears incapable of reacting with this receptor (note downward orientation of 'Y-hydrogens). The different configurations of the D-isomers (not shown) of ibotenic acid and kainic acid suggest that these compounds cannot combine with either the L-ibotenic receptor, or the receptor for L-aspartic acid. Also, when L-glutamic acid is maximally extended (Fig. 1), steric interference by the 'Y-hydrogens or, alternatively, the f3-hydrogens renders access of the molecule to the surface of the L-ibotenic acid receptor very difficult. Right: Folded conformations of D- and L-quisqualic acid, and a rotated L-a-kainic acid, demonstrate complementarity of these analogues to the receptor for L-aspartic acid (or folded glutamic acid).

9

10

N. T. Buu, E. PulL

AND

bicuculline combine in a folded conformation with the GABA receptor. Johnston et aJ. (1975) as well as Kier et aJ. (1974) suggest that the conformation required for the GABA receptor may be identical to that interacting with GABA aminotransferase. Although this cannot be ruled out, the evidence to date suggests that no direct relation exists between the affinity of GABA-like substances for the receptor, and for the enzyme (see "apparent affinity constants" K, Fig. 1; see also Buu & van Gelder, 1974). Finally, the preferred conformation of a substance in solution or in crystalline form has occasionally been used to indicate its orientation to the receptor. This argument may lead to spurious conclusions. It appears probable that the relative positions of the active centers in a receptor ultimately direct the conformational change in the substrate. No assumption can be made a priori that this change in substrate orientation represents that conformation, associated with the lowest calculated energy. TRUE ANTAGONISTS OF AMINO ACID ACTION

Theoretically, a true antagonist is one which, like the substrate(s) (agonist), binds to the receptor or close to it (allosteric). The antagonist subsequently prevents the agonist from inducing the response mediated by that receptor. At times, an antagonist may have ancillary actions of its own but these should not account for its antagonism. Several useful antagonists have been identified for the series of inhibitory amino acids. The action of glycine (or amino acids designated "glycine-like") can often be antagonized by strychnine, thebaine and brucine; GABA receptors (or the actions of amino acids designated "GABA-like") are less predictably blocked with bicuculIine, some can be blocked with picrotoxin and some with benzyl penicillin (c.f. Curtis & Johnston, 1974; Krnjevic, 1974). These agents which resemble glycine and GABA only to some extent, are not completely specific in their antagonism. For example, strychnine, which is considered to be a very reliable antagonist of the action of glycine, may itself sometimes cause a conductance change to K + (cf. Krnjevic, 1974). Another very interesting finding by Young & Snyder (1974) suggests that strychnine blocks the sites associated with the ionic conductance mechanism for chloride, which may be distinct from the glycine receptor proper. Despite some drawbacks, the above compounds exhibit remarkable specificity when compared to antagonists presently available for blocking the effects of excitatory amino acids, notably L-glutamic acid. No genuine competitive antagonist similar to D-tubocurarine for the nicotinic action of acetylcholine, has so far been found for the excitatory amino acids. Lysergic acid diethylamide (LSD-25) was the first substance found to "antagonize" excitation caused by either L-glutamate or 5-hydroxytryptamine

N. M.

VAN GELDER

in certain brain stem neurones (Boakes et al., 1970). The blockade in the case of L-glutamate is likely non-specific, and may be attributed to the important local anaesthetic activity of LSD-25 as demonstrated on peripheral nerve fibers (Di Carlo, 1961; see also Krnjevic & Phillis, 1963a). Many agents, in addition to local anaesthetics, exhibit in the CNS this unselective type of blocking effect on excitatory responses induced with L-glutamic acid and other putative neurotransmitters (cr. Curtis & Phillis, 1960; Krnjevic & Phillis, 1963a, 1963b; Glavinovic et al., 1974). Activation of neurones in the CNS with amino acids is prevented, without greatly altering the effect of acetylcholine, by L-methionine-DL-sulfoximine (Curtis et al., 1972; Haldeman & McLennan, 1972; Davies & Watkins, 1973a) and 2-methoxyaporphine (Curtis et al., 1972). Also, morphine has been reported to impair the excitatory action of L-glutamate (Dostrovsky & Pomeranz, 1973; Calvillo et al., 1974; Satoh et al., 1974). As above, the utilization of such compounds as antagonists is limited by their lack of selectivity but their blocking actions may contribute to the pharmacological effects that they produce in the CNS. A number of other substances which more closely resemble L-glutamic acid seem to be more specific in their blocking action. McLennan et al. (1971) demonstrated a reversible blockade of the excitatory action of L-glutamic acid by two related compounds: a-methyl-DL-glutamate and L-glutamic acid diethylester. These substances were reported to block glutamate-induced firing of thalamic neurones while the excitations caused by applications of acetylcholine or DL-homocysteic acid were relatively unaffected (Haldeman et al., 1972; Haldeman & McLennan, 1972). The blocking effect of a-methylDL-glutamate on glutamate receptors has not been confirmed in several regions of the CNS including the thalmus (Curtis et al., 1972), nor in the case of molluscan neurones (Kerkut et al., 1975). Yet, it is demonstrable on certain spinal neurones (Zieglgansgerger & Puil, 1973b; Padjen, 1974). The diethylester derivative, on the other hand, appears to block excitation produced by L-glutamate in many areas of the vertebrate CNS besides the thalamus (Curtiset al., 1972; Haldeman & McLennan, 1972; Dostrovsky & Pomeranz, 1973; Tebecis, 1973; Zieglgansberger & Puil, 1973b; Padjen, 1974). Not only is the action of acetylcholine much less affected by this agent, but L-glutamic acid diethylester also blocks excitation produced by L-aspartic acid and DL-homocysteic acid (Curtis et al., 1972). The ester is known to cause by itself, alterations in the resting potential and membrane conductance of spinal motoneurones (Zieglgansberger & Puil, 1973b; see also Szczepaniak & Cottrell, 1973). These findings require confirmation in view ofthe fact that some commercial preparations of L-glutamic acid diethylester appear to be seriously contaminated with L-glutamic acid (Wheal

Receptors for amino acids in excitable tissues & Kerkut, 1974; Roberts & Watkins, 1975). Stone (1973) has suggested moreover that some hydrolysis of the ester may occur which would make interpretations of results obtained with this compound even more difficult. Nevertheless, glutamate receptors at certain crayfish neuromuscular junctions can be reliably blocked with this agent (Lowagie & Gerschenfeld, 1974; Wheal & Kerkut, 1974) in the absence of detectable changes in membrane conductance, or without any effect on GABA receptors (Lowagie & Gerschenfeld, 1974). Several other g.lutamic acid esters possess similar blocking propertIes and some are in fact more potent than Lglutamic acid diethylester in blocking glutamate receptors at a crayfish neuromuscular junction (Lowagie & Gerschenfeld, 1974; Wheal & Kerkut, 1974). They are however inactive as antagonists in the vertebrate CNS (cf. Haldeman et al., 1972; Haldeman & McLennan, 1972, 1973). Antagonism of glutamate-excitation has been seen in several regions of the vertebrate CNS with I-hydroxy-3-aminopyrrolidone-2 (HA-966; Davies & Watkins, 1972, 1973a, 1973b; Curtis et al., 1973; Clarke et al., 1974). The excitation produced by acetylcholine at muscarinic receptors does not appear to be altered by this agent. The excitatory effects of L-aspartate (Davies & Watkins, 1972, 1973a) and DL-homocysteate on central neurones and Renshaw cell firing are reduced in addition to those of L-glutamate (Curtis et al., 1973). . Most of the substances which have been demonstrated to antagonize glutamate action in the CNS do not seem to be effective on molluscan glutamate re:c:ptors. On the other hand, DL-a-amino pimelic aCI~ has been reported to block inhibitory, but not eXCitatory responses evoked with application of L-glutamate (Kerkut et al., 1975). This compound and DL-a-aminoadipic acid are very similar to ~Iutamic acid .but have additional methylenic groups Incorporated In their chains. Application of DL-Qaminoadipic acid to molluscan neurones has been shown to cause only the initial, small depolarization of the biphasic response usually produced by Lglutamate application (Szczepaniak & Cottrell, 1973). Both analogues were previously noted to have very weak glutamate-like action onspinalneurones(Curtis & Watkins, 1960) and on insect muscle fibers (Usherwood & Machili, 1968). In summary, all compounds cited above appear !o fall in one of two categories. The first group Includes blockers of the depressant amino acids as well as substances which impair in a less specific way, the actions of excitatory amino acids; these agents bear only slight structural resemblance to the agonist which they antagonize. Members of the second class of "antagonists" are closely related to L-glutamate. In both classes, many of the compo~nds ~re not completely specific in their blocking actIOn since they also often produce agonistic effects.

11

CONCLUDING REMARKS

The above information suggests that the structure of an amino acid, the properties of its receptor and the !nteraction of the agonist with the receptor ar~ all very Important factors in determining the final response. One can demonstrate that certain modifications in the structure of these amino acids usually change the intensity but not the character of the response. In contrast, changes in conformation (or restrictions in conformational possibilities) may produce variations of either parameter and may sometimes permit a dis~inction to be made between two types ofreceptors which accept the same agonist. Finally, it is assumed with respect to the receptor, that its tertiary structure is ideally designed to accept the true substrate. At least two possibilities, therefore, may account for an ~nalogue inducing an enhanced effect. The first, an I?herent property of the analogue, is a higher affimty for the centers of the receptor which may be attributed to a difference in charge distribution (this may also contribute to slower removal of the agonist from the receptor). The second possibility is that the analogue may be more slowly removed from the receptors. An overall enhancement of a response to an amino acid does therefore not necessarily distinguish which of the two processes is occurring. According to the theory of absolute reaction rates, the rate of reaction (i.e. response) is proportional to t?~ concentration of an activated complex (or transition state) that is in equilibrium with the reactants (cf. Fig. 8, Lea & Usherwood, 1973a; Fig. 3, Oomura et al., 1974; see also Ziskind & Werman 1975). Thus, if a substance acts like the agonist ~ higher binding constant would be translated either by a faster rate of response (faster attainment of equilibrium) or an enhancement of the response (prolonged existence of equilibrium), or both. A slower inactivation of the complex, on the other hand, may enhance the response but should not be reflected by an increase in the rate of the response. Clearly then, such distinctions can only be made under conditions in which the substrate concentration remains in excess during the total time of obs~rvation (i.e. initial rates, steady-state approximatIOn and substrate saturating conditions; Briggs & Haldane, 1925; Cleland, 1963; MaWer & Cordes, 1966, chapter 6). Acknowledgements-We are grateful to Mr. R. Pe!oquin of th~ photography department for lending his skill m rendermg the Dreiding models in three dimensional perspective. This manuscript was made possible by grants from the Medical Research Council of Canada. REFERENCES

AMES A., TSUKADA Y. & NESBETT F. B. (1967) Intracellular CI-, Na +, K +, Ca2+, Mg2+ and Pinnervous tissue; response to glutamate and to changes in extracellular calcium. J. Neurochem. 14, 145-159.

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N. T. Buu. E. PulL AND N. M. VAN GELDER

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