Excitatory amino acids: physiological and pharmacological probes for neuroscience research

Haruhiko Shinozaki and Michiko Ishida

The Tokyo Metropolitan Institute of Medical Science, 3-1 8-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan

Abstract. The 2S,3S,4S-isomer (L-CCG-I) of 2-(carboxycyclopropyI) (CCG) is a potent metabotropic agonist. L-CCG-I depressed monosynaptic excitation in the newborn rat spinal motoneurone at low concentrations well below those causing postsynaptic depolarization. 2~,3~,4~-CCG (L-CCG-IV) is a potent N-methyl-D-aspartate (NMDA)-type agonist. In cultured rat hippocampal neurones, L-CCG-IV caused marked increase in intracellular ca2+concentrations. 6-Carboxylated L-CCG-IV (DCG-IV), which is a tricarboxylated CCG derivative containing both chemical moieties of L-CCG-I and L-CCG-IV, depressed preferentially monosynaptic excitation of spinal reflexes in lower concentrations than L-CCG-I. 4-(2-Methoxypheny1)-2-carboxy-3- pyrrolidineacetic acid (MFPA), which is the most potent kainoid yet described, is superior to acromelic acid A in causing depolarization of the newborn rat spinal motoneurone. In addition to MFPA, some non-kainoids demonstrated considerably high depolarizing activities. These new compounds would Address for correspondence: provide useful probes for neuroscience research. Haruhiko Shinozaki, The Tokyo Metropolitan Institute of Medical Science, 3- 18-22. Honkomagome,- Key words: L-glutamate, NMDA, kainate, excitatory amino acids, Bunkyo-ku, Tokyo 113, Japan metabotropic glutamate receptors, spinal reflex, transmitter release 44 H. Shinozaki and M. Ishida INTRODUCTION acids of natural origin are described. These com- pounds provide useful information about the mech- L-Glutamate has been believed to be a major ex- anism underlying glutamate's transmitter function. citatory neurotransmitter at many synapses in ver- tebrates and invertebrates. The study of excitatory amino acids began with the investigation of their 2-(CARBOXYCYCLOPR0PYL)- structure-activity relationships. At present, the GLYCINE pharmacological study of excitatory amino acids is progressing rapidly toward elucidation of the physi- The glutamate analog, 2-(carboxycyclopro- ological roles of each receptor subtype. Central ex- py1)glycine (CCG), was isolated from the plants citatory amino acid receptors now are most Aesculuspawij'lora and Blighia sapida (Fowden et conveniently subdivided into five main classes al. 1969). CCG is a conformationally restricted (Monaghan et al. 1989); N-methyl-D-aspartate analog of glutamate, in which the cyclopropyl (NMDA)-, kainate-, a-amino-3-hydroxy-5-methyl- group fixes the glutamate chain in an extended or 4-isoxazolepropionic acid (AMPA)- and L-2- folded form (Kurokawa et al. 1985, Yamanoi et al. amino-4-phosphonobutyric acid (L-AP4)-type 1988) and CCG has eight diastereomers (Shimamo- receptors, and metabotropic glutamate receptors to et al. 1991). Therefore, CCG would provide use- which are coupled to inositol-l,4,5-triphosphate ful information about the interaction between the (IP3) and diacylglycerate turnover. NMDA recep- conformation of glutamate molecules and activa- tors are competitively and effectively blocked by a tion of each receptor subtype. Eight CCG stereo- number of a-phosphono-a-amino acids, notably isomers (Fig. 1) demonstrated a large variety of D-(-)-2-amino-4-phosphonovaleric acid (D-APV) depolarizing activities in the isolated newborn rat and 3-(2-carboxypiperazin-4-y1)propyl-1 -phosphonic spinal cord (Shinozaki et al. 1989b, c). The depola- acid (CPP). Some derivatives such as 6- rizing activity of the 2S,3S,4S-isomer (L-CCG-I, an cyano-7-nitroquinoxaline-2,3-dione (CNQX), extended form) is higher than that of L-glutamate in 6,7-dinitroquinoxaline-2,3-dione(DNQX) and 2,3- the newborn rat spinal motoneurones, and is almost dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxali equal to that of trans-ACPD. Neither selective ne (NBQX) are blockers of both AMPA- and kai- NMDA blockers nor CNQX depressed depolariz- nate-type receptors, but highly selective and potent ing responses to L-CCG-I, demonstrating a clear antagonists at non-NMDA-type receptors are not preference for non-NMDA, non-kainate and non- yet discovered. The 1S, 3R-enantiomer (1S,3R- AMPA receptors. Depolarizing responses to L- ACPD) of 1-aminocyclopentane-trans- 1,3-dicar- CCG-I and trans-ACPD were markedly decreased boxylate (trans-ACPD) was reported to be a by reducing the temperature of the perfusing fluid, selective agonist at metabotropic glutamate recep- clearly differentiating between ionotropic type tors (Irving et al. 1990). Although selective anta- agonists and the others (Ishida et al. 1990). These gonists are essential for the pharmacological study responses may suggest the participation of metabo- of neurotransmitters, specific and potent glutamate tropic glutamate receptors in the L-CCG-I-induced agonists are still required to elucidate glutamate depolarization of newborn rat spinal motoneurones. function further. As standard in such study, some Activation of metabotropic glutamate receptors compounds of natural origin, such as , blocks the slow ca2+-dependent K+ conductance , and , and increases the membrane excitability of neur- have played key roles as valuable pharmacological ones (Schoepp et al. 1990, Anwyl 1991, Baskys probes for neuroscience research on excitatory 1992). L-CCG-I induced oscillatory responses in amino acids. In the present paper, neuropharmaco- Xenopus oocytes injected with rat brain mRNA logical actions of some novel excitatory amino (Ishida et al. 1990), and stimulated inositolphos- Excitatory amino acids 45

Metabotropic Glutamate uptake NMDA-type, agonist glutamate receptor inhibitor agonist 1 C02H

H02C I bCo2.J H NH2 NH2 H NH2 I

L-CCG-I L-CCG-I1 L-CCG-111 I L-CCG-IV : I )------.I I I

I I D-CCG-I / D-CCG-11 D-CCG-111 D-CCG-IV : I ------J------i potent

Quisqualate > D-11 > L-IV Kainate > NMDA > D-lV > L-1 >> L-Glutamate > L-Ill

Fig. 1. Chemical structures of eight diastereomers of 2-(carboxycyclopropy1) glycine. phate formation in rat hippocampal synaptoneuro- times when monosynaptic excitation was de- somes, demonstrating that L-CCG-I was a potent creased. Therefore, L-CCG-I seems to be function- agonist for metabotropic glutamate receptors which ally dissimilar to 1S,3R-ACPD. are linked to GTP binding proteins (Nakagawa et al. The 2S,3S,4R-isomer (L-CCG-111, a folded 1990). form) caused marked potentiation of depolarizing Metabotropic glutamate agonists block the exci- responses to L-glutamate, D- and L- aspartate more tatory synaptic transmission supported by the iono- effectively than L-(-)-threo-3-hydroxyaspartate, tropic glutamate receptor, and may therefore play a but did not affect the depolarization induced by critical role in synaptic plasticity (Anwyl 1991). L- kainic acid, quisqualic acid, NMDA and D-gluta- CCG-I preferentially reduced monosynaptic dis- mate (Shinozaki et al. 1989~).There is clear evi- charges induced by electrical stimulation of dorsal dence that this is due to inhibition of uptake of some root fibres of newborn rats in low concentrations excitatory amino acids (Kawai et al. 1992). In con- well below those causing postsynaptic depolariza- trast to the actions of the 2S,3R,4S-isomer (L-CCG- tion (Fig. 2), though both mono- and poly-synaptic IV, a folded form), which is a potent NMDA agonist discharges were depressed in high concentrations (see below), L-CCG-I11 demonstrated considerably (Shinozaki and Ishida 1992). This reduction of lower depolarizing activity despite a folded com- monosynaptic discharges was neither depressed mon glutamate structure. by GABA antagonists such as picrotoxin and 2- Among eight CCG stereoisomers, the 2R,3S,4S- hydroxysaclofen nor by any other pharmacological isomer (D-CCG-11) showed the highest depolariz- agents including glutamate antagonists. lS,3R- ing activity - about 5 times higher than that of ACPD also depressed spinal reflexes, but it was ac- NMDA in the newborn rat spinal cord - followed by companied by postsynaptic depolarization at all L- CCG-IV (Shinozaki et al. 1989b). D-CCG-I1 and 46 H. Shinozaki and M. Ishida

A b

,LAI ~(~~~~~~~~(~~LLLL~~LL~~~&LLLL~~~ mv L-CCG-I 1 pM L-CCG-I 3 pM 2 min Fig. 2. Preferential depression of B monosynaptic excitation of spi- - a nal reflexes by L-CCG-I in new- b c J2 mv born rats. A, the upper trace 20 ms represents low frequency com- -. -. ponents of spinal reflexes induced JY--I 'j~--7 1 by the electrical stimulation of I I the dorsal root fibres. R, sample C records of spinal reflexes on a X- 5= Y recorder. a, b, c, d correspond -E to those in A. C ,The time course 5 of the peak amplitude of monosy- 8a -.- naptic excitation in the presence aE" of L-CCG-I. 0 L-CCG-IV are potent and specific NMDA agonists. closely mimics the folded conformation of L-gluta- This is the first time to find that an NMDA agonist mate, therefore, an active conformation of L-gluta- with an L-configuration is more potent than mate at NMDA receptors is considered to be a NMDA. It is reasonable that L-CCG-IV causes an folded form. increase in intracellular ca2+ concentrations, be- The L-CCG-IV derivatives, 6R- and 6s-meth- cause ca2+ influx into neurones through receptor oxymethyl-CCG and 6R-benzyloxymethyl-CCG, ionchannels is one of characteristic features of func- were much more potent than L-glutamate in causing tion of NMDA receptors. ca2+ may play a critical depolarization of newborn rat spinal motoneurones. role in causing excitotoxic neuronal damage 6R-Methoxymethyl-CCG was approximately twice (Garthwaite and Garthwaite 1986, Choi 1987). L- as potent as 6s-methoxymethyl-CCG and about 10 CCG-IV increased intracellular ca2+ concentra- times more potent than 6R-benzyloxymethyl-CCG. tions much more markedly than NMDA in the The depolarization induced by 6R-derivatives was cultured rat hippocampal neurone loaded with fura- completely depressed by CNQX, but was almost in- 2 (Shinozaki et al. 1991a, Kudo et al. 1991). The sensitive to CPP (Ishida et al. 1991). The 6R-sub- potency of L-CCG-IV to cause the increase was stituted CCG is a non-NMDA receptor agonist about 10 times higher than that of L-glutamate, and despite the fact that L-CCG-IV and its 6R-sub- about 300 times higher than that of NMDA, and D- stituted compound have a folded common gluta- CCG-I1 and the 2R,3S,4R-isomer (D-CCG-IV) mate structure with the same configuration and showed almost equal potencies to L-glutamate. conformation. Other CCGs were much less potent than NMDA. L- In the isolated dorsal root C-fibres of immature CCG-IV induced marked neuronal death in the CAI rats, kainic acid, domoic acid and L-glutamate pro- area but not in the CA3 area of the rat hippocampus duced considerable depolarization in a dose de- when injected intraventricularly, corresponding to pendent manner, but quisqualic acid and AMPA the distribution of NMDA receptors. The carbon caused only a slight depolarization at considerably chain of glutamate can conform almost completely high concentrations, and NMDA did not cause any to that of CCG, and the structure of L-CCG-IV depolarization even in high concentrations Excitatory amino acids 47

(Agrawal and Evans 1986, Shinozaki et al. 1991b). Therefore, the dorsal root fibre of the immature rat HOOC, CH spinal cord is practically useful for pharmacological POoH HOOC-& 1CH-cH classification of agonists. 6R-Sub- , NHZ HOOC stituted CCGs caused a considerable depolarization in relatively low concentrations, suggesting the 6R- substituted CCGs are kainate agonists (Ishida et al. Fig. 3. Chemical structure of DCG-IV. 1991). L-CCG-IV caused slight depolarization of depolarizing activity became lower than that of L- the dorsal root fibre at high concentrations. Signifi- CCG-IV or L-CCG-I, and the depolarization cantly high concentrations of L-CCG-I11 induced evoked by DCG-IV was completely depressed by only a slight depolarization, but other CCGs and 6s- selective NMDA antagonists, unlike the depo- methoxymethyl-CCG, which showed NMDA-like larization caused by L-CCG-I. As previously depolarization in the spinal motoneurone, did not mentioned, L-CCG-I depressed preferentially cause any depolarization of the dorsal root fibre monosynaptic excitation of the rat spinal reflexes. even at high concentrations. DCG-IV depressed monosynaptic excitation more effectively than L-CCG-I in low concentrations 2-(47 6~D1CARBoXYCYCLoPRoPYL) well below those causing postsynaptic NMDA-type GLYCINE depolarization. Baclofen, a GABAB agonist, effec- tively depresses spinal reflexes in this preparation, DCG-IV (Fig. 3) is a tricarboxylated CCG but the activities of DCG-IV to depress the mono- derivative containing both chemical moieties of synaptic excitation was much higher than that of ba- L-CCG-I and L-CCG-IV. DCG-IV caused NMDA- clofen (Fig. 4). Picrotoxin, 2-hydroxysaclofen, type depolarization of spinal motoneurones but its bicuculline, and other pharmacological agents did

Baclofen

Concentration (M)

Fig. 4. Dose response curves for DCG-IV, baclofen, L-CCG-I and L-AP4 in inhibiting monosynaptic excitation of spinal rei- lexes induced by the electrical stimulation of the newborn rat dorsal root fibres. The inhibitory ratio of monosynaptic excitation was plotted against the concentration of each compound. 48 H. Shinozaki and M. Ishida not block the reduction of monosynaptic reflexes acid or domoic acid in newborn rat spinal moto- induced by DCG-IV. Furthermore, DCG-IV itself neurones (Ishida and Shinozaki 1988, Shinozaki et did not depress any type of depolarization induced al. 199lb), and its electrophysiological properties by excitatory amino acids. lS,3R-ACPD and L- are quite similar to those of kainic acid or domoic AP4 also depressed monosynaptic discharges, but acid (Shinozaki 1988, Shinozaki 1992). Therefore, DCG-IV was functionally dissimilar to L-AP4, it is reasonable to assume that acromelic acids show lS,3R-ACPD, L-CCG-I or baclofen. These inhibi- excitotoxicity quite similar to kainic acid. However, tory actions of DCG-IV on monosynaptic excitation a chain of abnormal behavioral signs induced by seem to be due to activation of presynaptic recep- systemic administration of acromelic acid A to the tors. rat was quite distinct from that of systemic kainate DCG-IV is expected to be a metabotropic gluta- or domoate. The most pronounced changes were mate receptor agonist from the structural similarity tonic extension of the hindlimbs, followed by flac- between DCG-IV and L-CCG-I. L-CCG-I stimu- cid paralysis and persistent spastic paraplegia lates inositolphosphate formation and decreased (Shinozaki et al. 1989a, 199 1b, Kwak et al. 1992b). forskolin-stimulated cAMP content, however, These changes were never caused by kainic acid, in- DCG-IV does not increase phosphatidylinositol stead, it caused severe limbic motor seizures. Sys- turnover, instead, preferentially inhibits forskolin- temic acromelic acid A causes selective neurone stimulated cAMP formation in the nerve cell at con- damage of small neurones in the lower spinal cord, siderably lower concentration. but it does not cause lesions of spinal motoneurones (Kwak et al. 1992b). KAINATE AGONISTS Differences in behavioral signs and the distribu- tion of neurone damage between acromelic acid A From the poisonous mushroom Clitocybe ac- and kainic acid led us to search for novel kainoids romelalga, three kainoids (acromelic acid A, B and which possibly revealed different kinds of neu- C) were isolated, which possess a constitutional ropharmacological actions (Fig. 5) (Ishida and moiety of kainic acid (Konno et al. 1983,1988,Fushiya Shinozaki 1991, Shinozaki 1992). Some kainate et al. 1990). Acromelic acid C is not an isomer of derivatives caused depolarization much more effec- acromelic acid A or B, but has a structure of decar- tively than kainic acid. In newborn rat spinal boxylated acromelic acid B. Acromelic acid A motoneurones, 4-(2-methoxypheny1)-2-carboxy- caused a more marked depolarization than kainic 3-pyrrolidineacetic acid (MFPA) was the most po-

H

8 ,,~--COOH ,,, -COOH Hoocyf,,,-COOH

QcooHH [888 GcooH QcooH H HOOC = H Kainic acid Domoic acid Acromelic acid A

o8OCH3\,,-COOH 88,-COOH Fig. 5. Chemical struc- tures of kainic acid, do- QcmH QcooH moic acid, acromelic acid and newly synthesized potent kainoids. MFPA HFPA Excitatory amino acids 49 tent among test samples in causing the depolariza- kainic acid or domoic acid. However, acromelic tion. 4-(2-Hydoxypheny1)-2-carboxy-3-pyrrolidi- acids showed a considerably lower affinity for kai- neacetic acid (HFPA) also showed considerably nate receptors than kainic acid. Of particular inter- high depolarizing activity. The rank order of depo- est, acromelic acid A and MFPA possessed valuable larizing activities in motoneurones was MFPA > ac- affinities for AMPA receptors. Smith (1992) recent- romelic acid A > domoic acid 2 HFPA 2 acromelic ly reported that, in the absence of potassium thio- acid B > HMPPA = CPPA = kainic acid > MPPA cyanate, acromelic acid A is the most potent = FPA > CNOPA > MKPA. As acromelic acid A is displacer of AMPA binding yet described, and he so far the most potent among known excitatory has presented that acromelic acid A distinguishes amino acids, note that MFPA is more potent than two kainate binding sites in rat brain synaptic plas- acromelic acid A. The actions of MFPA and HFPA ma membranes. These compounds are expected to were effectively depressed by CNQX, but not by se- provide useful information for elucidating the lective NMDA blockers. The rank order of above diversity of kainate receptor function. kainoids in the isolated dorsal root fibre is domoic At this time the above mentioned discrepancy acid > acromelic acid B > 5-bromowillardiine 2 has no clear-cut explanation, though the idea of MFPA > acromelic acid A > HFPA > kainic acid > more than two types of kainate receptor subtypes is CPPA > HMPPA > CNOPA = FPA 2 MKPA = L- quite appealing. Systemic administration of MFPA glutamate. This order differed considerably from induced interesting behavioral signs in rats, includ- that obtained in spinal motoneurones. Cross desen- ing both tonic extension of the hindlimbs and limbic sitization between kainic acid and above kainoids seizures, which are characteristic of acromelic acid suggests that these kainoids activated receptors in A and kainic acid, respectively. Multiplicity of kai- common with kainic acid in the dorsal root fibre. nate receptors also is proposed on the basis of elec- The rank order of ICso values for displacement trophysiological evidence from cultured rat 3 with high affinity to [ HI kainate binding sites in the hippocampal neurones (Iino et al. 1990). In addi- adult rat spinal cord was domoic acid > HFPA tion, recent studies on cDNA for glutamate recep- kainic acid = MFPA > quisqualate > acromelic acid tors suggest the presence of a family of kainate B > L-glutamate > acromelic acid A >> NMDA = receptor subunits with regional difference in the rat AMPA, and that of [3~]~~~~was quisqualate > brain. Kainate or AMPA receptors may be com- AMPA > MFPA = acromelic acid A > L-glutamate posed of heterooligometric subunits. The associ- > HFPA > domoic acid > kainic acid >> NMDA ation of these subunits with one another would (Kwak et al. 1992a). MFPA and HFPA demon- provide a number of different complexes, each ex- strated high binding affinities for kainate receptors hibiting distinct electrophysiological properties in the rat spinal cord, substantially comparable to such as preferential response to various kainate

0 COOH

Br CH30 /O

Fig. 6. Chemical structures of non-kainoids that activate kainate receptors. 50 H. Shinozaki and M. Ishida

agonists. Experimental results that non-kainoids ac- electrophysiological actions of a conformationally re- tivate kainate receptors led us to search for new stricted glutamate analogue in the rat spinal cord and Xenopus oocytes. Brain Res. 537: 3 11-314. non-kainoid kainate receptor agonists. So far 5- Ishida M., Shimada Y ., Shimamoto K., Ohfune Y ., Shinozaki bromowillardiine has been known as a non-kai- H. (1991) Changes in preference for receptor subtypes of noids potent excitant of the dorsal root fibres. In configurational variants of a glutamate analog: conversion fact, as mentioned above, 5-bromowillardiine was from the NMDA-type to the non-NMDA type. Brain Res. superior to MFPA or acromelic acid A in causing 550: 152-156. Ishida M., Shinozaki, H. (1988) Acromelic acid is a much depolarization of the dorsal root fibre. The com- more potent excitant than kainic acid or domoic acid in the pounds shown in Fig. 6 demonstrated depolarizing isolated rat spinal cord. Brain. 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