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Spinal glutamate receptors
Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.
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Spinal glutamate receptors David Lodge and Ann Bond Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom
INTRODUCTION Glutamate, and possibly other acidic amino acids, are now accepted as the major mediators of excitatory neurotransmission in the brain and spinal cord. The initial discovery of glutamate's stimulatory effects on neurones of the central nervous system (CNS) stems from work in the 1950s 1,2]. Despite its ubiquitous function in general cellular metabolism, the transmitter role of glutamate was slowly accepted during the 1960s and 70s. This was aided by separate but linked observations in pioneering laboratories. 1. Neurones in different parts of the brain and spinal cord showed different sensitivities to derivatives of glutamate [3,4]. These included synthetic derivatives such as N-methyl-D-aspartate (NMDA) and natural products such as quisqualate and kainate [5]. 2. Secondly, other synthetic derivatives were found to have antagonistic actions against some of the above glutamate analogues and against synaptic excitations in the CNS [6-9]. These included 1-hydroxy-3-aminopyrrolidone-2 (HA-966), D-a-amino-adipate, ~/-glutamyl-amino-methyl-sulphonate (GAMS) and glutamate diethyl ester (GDEE). The former two selectively reduced responses to NMDA, a pharmacological feature which was shared with several divalent cations, such as magnesium [10]. GAMS and GDEE were shown to have selective effects versus quisqualate and kainate induced responses respectively. Additionally, short latency monosynaptic responses were reduced by the quisqualate antagonists whereas longer latency polysynaptic responses were reduced by NMDA antagonists both in vitro and in vivo [11-13]. 3. Neurochemical studies using radiolabelled glutamate and some of the above analogues showed specific binding sites and uptake mechanisms [3, 14, 15]. The presence of these presumed postsynaptic receptors and transporter systems provided key support for the electrophysiological evidence for the transmitter role of L-glutamate. The selectivity of many of these early agonists and antagonists was poor but nevertheless a concept of three glutamate receptor subtypes developed [7, 8, 11, 16, 17], and despite close examination over the years, this subdivision still holds generally true today (see below). NMDA receptors. The NMDA receptor has received considerable attention, largely because of the early development of selective tools for its study. NMDA itself is a specific ligand for this receptor with little action at other types of glutamate receptor. In particular, the discovery of a highly selective and competitive NMDA antagonist
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in D-2-amino-5-phosphonopentanoate (AP5) [17, 18] allowed many aspects of the physiological and pathological role of this receptor type to be elucidated. The discovery that arylcyclohexylamines such as ketamine and phencyclidine and benzomorphans such as cyclazocine and N-allyl-normetazocine (SKF10,047) were non-competitive channel-blocking NMDA antagonists, was particularly helpful for whole animal electrophysiological and behavioural studies, since these compounds, unlike the competitive antagonists, crossed the blood-brain barrier quickly [19,20]. Three other key discoveries provided insights into the physiology of the NMDA receptor. Firstly, the block of the NMDA receptor by magnesium [10] was shown to provide the key voltage-dependent property of this receptor [21]. At resting membrane potentials around -70 to -60 mV, normal extracellular levels of magnesium provide a brake on the permeability of the receptor, but as neurones depolarise due to synaptic inputs (or to metabolic challenges) the magnesium braking action is relieved and the channels conduct current freely. Secondly, the NMDA receptor was shown to be highly permeable to calcium [22]. The resulting increase in intracellular calcium is important not only for physiological, but also for pathological, facilitation of calcium dependent enzymic functions. Thirdly, allosteric modulation of NMDA receptors by glycine acting at a separate recognition domain on the NMDA receptor complex [23]. It is now known that the NMDA receptor requires co-activation of both the glutamate and glycine recognition sites and indeed a co-transmitter role for glycine has been proposed. In the presence of low amounts of glycine the NMDA receptor appears to undergo desensitisation which can be reversed by increasing the external concentration of glycine [24, 25]. It is still a matter of some debate as to whether extracellular levels of glycine are sufficient to fully saturate this site. Nevertheless because of the requirement for co-activation of the glycine site, considerable pharmacological effort has been made to produce partial agonists and full anatgonists at this receptor [25]. Alongside these largely electrophysiological studies, others showed that NMDA receptors mediated profound neurotoxicity [26-30] and have a central role in epileptiform discharges [31-32] in vitro and in vivo. As a result, the therapeutic potential of NMDA receptor antagonists has emerged and several agents are under development by pharmaceutical companies [33]. The first NMDA receptor subunit (now known as NMDA-R1) was cloned and expressed as a functional NMDA receptor channel complex by Moriyoshi, Nakanishi and colleagues [34]. By homology cloning, NMDA-R2 subunits, A,B,C and D, were also identified. NMDA-R2 subunits, unlike NMDA-R1, do not, however, form functional homomeric channels. It has subsequently been shown that native NMDA receptors consist of heteromeric complexes of NMDAR1 associated with NMDA-R2A-D, presumably in a pentameric structure. All such known NMDA receptor configurations have pharmacology in general agreement with that described above, although variations in kinetics and pharmacology have been reported. Constructs with NMDA-R2C are relatively insensitivite to glycine and to Mg 2+, which may correlate with the pharmacology of NMDA receptors of the cerebellum where 2C subunits are highly expressed. As NMDA receptors are not the major subject of this chapter, readers are referred to recent reviews of their molecular biology and pharmacology [13, 3537].
243 AMPA and kainate receptors. Until recently there has been a lack of pharmacological tools for other glutamate receptors. The original subdivision of non-NMDA receptors depended on the agonist selectivity of quisqualate, which has subsequently been shown also activate phospholipase-coupled metabotropic receptors [38]. The synthesis of a new and more selective ligand was therefore a crucial step. The discovery of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) as a GDEE sensitive agonist [39], led to the re-naming of the non-NMDA receptors as AMPA and kainate receptors [16, 40]. Furthermore radioligand binding experiments have allowed separate high affinity [3H]AMPA and [3H] kainate binding sites to be distinguished [14, 40, 41]. Some cross displacement, particularly of [3H]AMPA by unlabelled kainate, however, confirmed electrophysiological observations of some non-selectivity of these two agonists, which still remains a problem. The characteristic pharmacology of AMPA was subsequently corellated, following the use of patch clamp techniques, with its kinetic profile; thus, AMPA evoked rapidly desensitising responses in central neurones whereas those to kainate were relatively non-desensitising [42, 43]. Block of AMPA currents, however, by prior application of kainate suggested a common site but with different mechanisms, AMPA acting as a partial agonist and kainate as a full agonist [42, 44-46]. On this basis, the separation between kainate and AMPA receptors was unlikely to be achieved without other selective agents or molecular biological identification of distinct receptor subunits. Both these advances have been made in the last five years.
Competitive AMPA receptor antagonists. The low margin of selectivity between AMPA and kainate responses with the early antagonists such as GDEE and GAMS especially in in vitro studies required the development of more potent and selective competitive antagonists. The first such compounds showing selective AMPA antagonist activity were the 6-cyano-7-nitro- and 6,7-dinitro-quinoxalinediones, CNQX and DNQX, [47] but their selectivity for AMPA over kainate was not more than five-fold. A further synthetic development led to 3-dihydroxy-6-nitro-7-sulphamoyl-benz(F)quinoxaline (NBQX) which has a 30-fold greater selectivity at displacing the binding of [3H]AMPA, rather than that of [3H]kainate [48]. NBQX also has similar 30 fold higher potency as an antagonist of AMPA-evoked depolarisations on cortical slices in vitro [48, 49]. In a later development of competitive AMPA receptor antagonists, LY293558, a decahydroisoquinoline with a tetrazole group substituted at the 6 position, was shown to selectively displace [3H]AMPA binding on rat brain membranes and to antagonise AMPA-induced depolarisations on cortical slices and, following systemic administration, to block central effects of AMPA receptor agonists [50]. This compound is also interesting from a medicinal chemistry stand-point. In LY293558 the decahydroisoquinoline is separated from the tetrazole by 2 carbons [50]; a single carbon in this chain yields a selective NMDA antagonist, LY233536 [51] whereas a direct carbon linker gives a non-selective NMDA and AMPA antagonist, LY246492 [52].
244 Similar data to that with LY293558 has been reported for two analogues of AMPA called AMOA and AMNH which respectively are substituted with a 3carboxymethoxy and a 2-methylisoxazole on the isoxazole ring of AMPA [53]. AMOA selectively displaces [3H]AMPA binding whereas AMOA displaced [3H]AMPA and low affinity [3H]kainate binding more or less equally; neither compound displaced high affinity [3H]kainate binding. On cortical slices AMOA and AMNH were weakly selective for AMPA- and kainate- induced depolarisations respectively [53]. AMPA r e c e p t o r channel blockers A number of invertebrate polyamine toxins have recently been shown to be open channel blockers of glutamate receptors initially at the neuromuscular junction of locusts [54] and recently on central mammalian neurones [54-57]. These toxins include argiotoxin 636, Joro spider toxin and philanthotoxin. Although these polyamine toxins block NMDA receptors at least in some preparations, in others they also block responses mediated by AMPA receptors more selectively [58-60].
Allosteric modulators of AMPA receptors. In addition to the above competitive antagonists, a 2,3-benzodiazepine, GYKI 52466 [61-63] has highlighted a new mechanism of modulating non-NMDA receptors in a non-competitive manner. GYKI 52466, selectively reduces responses to AMPA and not to NMDA, and is about one fifth as potent against kainate responses, on rat cortical slices [63]. This is, therefore, another useful tool for separating between AMPA and kainate receptors. LY300164 (GYKI 53655), the 3-methyl-carbamoyl derivative of GYKI 52466, is approximately 10 times more potent than GYKI 52466 on AMPA receptors but less potent on kainate receptors [64, 65]. As desensitisation is a prominent feature of AMPA receptors, a plant lectin, concanavilin A, previously shown to block desensitisation of insect glutamate receptors [54], was tested and found to enhance AMPA and kainate responses on hippocampal [43, 66], retinal [67] and dorsal root ganglion (DRG) [68] neurones. More recently, a series of benzothiazides including diazoxide and cyclothiazide, were shown to reduce glutamate receptor desensitisation and hence increase AMPA responses on hippocampal [69, 70] and cortical [71] neurones. As will be discussed below, it has become clear that concanavilin A and cyclothiazide act selectively on rapidly inactivating kainate and AMPA responses respectively. Molecular biology of AMPA and kainate receptors. An AMPA receptor, GluR1 (or GluRA) was the first glutamate receptor to be cloned and expressed [72]. Our concepts of AMPA and kainate receptors have been revolutionised over the past six years through the application of molecular biology techniques to this area. In the ensuing five years, the total number of non-NMDA receptor subunits has increased to nine. They can be divided into two major types: a) the AMPA receptors, GluR1-4, which bind AMPA with high affinity and have a rapidly desensitising response to this agonist and a smaller but non-desensitising response to kainate. These channels with the exception of GluR2 are permeable to calcium [73]. In the proposed TM1 region of GluR2 an
245 arginine occurs at the site at which glutamine is positioned in GluR1,2 and 4. This so called Q/R site has been shown by site directed mutagenesis studies to be a major determinant of calcium permeability [35-37]. b) the kainate receptors, GluR5-7 and KA1-2 which bind kainate with medium and high affinity respectively [35-37] Only GluR5-6 form homomeric channels whereas the others require heteromeric expression with GluR5 or 6 to become functional. GluR5 and 6 also have the Q/R site in the TM2 region and in this case editing appears to be variable throughout the CNS [35-37]. Only a small part of the molecular biology will be covered here; more comprehensive reviews are recommended [35-37, 74, 75]. Although the molecular pharmacology of glutamate receptors is still in its infancy, some interesting data are emerging:1. The calcium permeability of non-NMDA receptors is controlled largely by the so called Q/R site in the putative second transmembrane segment (TM2). This site is occupied by glutamine (Q) in GluRsl, 3 and 4 but by an arginine (R) in GIuR2. This arginine accounts for the low permeability to calcium of both homomeric or hetromeric channels with GluR2. The equivalent site in the NMDA receptors is occupied by an asparagine which may account for the calcium permeability and magnesium block of the NMDA channel. The genomic DNA codes for a glutamine at this Q/R site even in GluR2; so editing of RNA (CAG to CIG by adenosine deaminase) results in an arginine in the GluR2 protein. 2. Desensitisation is controlled in part by alternative splicing of the so-called flip and flop region in the loop between TM3 and TM4 [76]. The presence of glycosylation sites [77-79] and at least part of the agonist binding domain [80] in the same loop suggests its extracellular location. 3. The topology of the glutamate receptor as a result of such studies with m u t a n t receptors is now thought to be quite different from the four transmembrane crossing model suggested for nicotinic receptors, etc. [74,77-79]. TM2 is thought to loop within the membrane rather than crossing it, which in turn leads to an extracellular TM3-4 loop and an intracellular carboxy terminus. The Q/R site is thought to be near the apex of the intramembrane TM2 loop. This model, which has similarities with voltage-dependent ion channels, highlights the limitations of interpretations based solely on hydrophobicity plots. 4. NBQX and CNQX are relatively non-selective antagonists across all the recombinant non-NMDA receptors. Thus NBQX blocks GluR1-4 receptors in the 100nM region [81, 82] and GluR5 and 6 in the 2mM region [83, 84]. 5. By contrast LY293558, tested on human recombinant receptors, displaces GluR1-4 binding with 2-40uM potency, and 900nM potency on GluR5 but is almost inactive (mM) on GluR6 (R.J. Kamboj - unpublished observations). This selectivity is also seen in patch clamp studies [84]. Thus LY293558 could be used to differentiate between GluR5 and GluR6 based kainate receptors, although the situation in GluR6 heteromerics is unkown. 6. GYKI 52466, and other more potent 2,3-benzodiazepines, are active on GluR1-4 with only small effects on the kainate preferring subtypes. Thus, LY300164 (GYKI 53655) is effective at lmM as an antagonist of recombinant GluR1-4 and native AMPA receptors whereas 100uM is required to block native and recombinant kainate receptors (64,65, D. B l e a k m a n unpublished observations).
246 7. Interestingly the two blockers of desensitisation, cyclothiazide and concanavilin A, show complimetary selectivities. Cyclothiazide blocks the desensitisation on recombinant GluR1-4 but, if anything, reduces responses on on kainate receptors, GluR5 and 6 [85]. Concanavilin A on the other hand markedly potentiates responses on these kainate receptors but is only weakly effective on the recombinant AMPA receptors [85]. Such results parallel those from the GluR1-4-dominated hippocampal neurones and the GluR5-dominated dorsal root ganglion cells [64, 68, 86, 87] and show the usefulness of these compounds in differentiating between AMPA and kainate receptors. 8. Argiotoxin 636, Joro spider toxin and philanthotoxin blocks AMPA receptors GluR1, 3 and 4 but is much less active on the homomeric or heteromeric channels containing GluR2 and GluR6 [88-90]. GluR6 may also be edited to an arginine at the Q ~ site. Hence this differential effect of toxins has been related to the Q/R site editing of GluR2 and unedited, glutaminecontaining, versions of GluR2 and GluR6 are sensitive to Joro spider toxin. Similarly conversion to an arginine at the Q/R site of GluR4 renders it insensitive to argiotoxin. So the two properties of calcium permeability and toxin efficacy appear to be controlled at least partially by the molecular entities in the channel.
Metabotropic glutamate receptors.
In addition to ion channel coupled receptors, glutamate also activates second messenger systems via G-protein coupled receptors. The molecular biology and pharmacology of metabotropic glutamate receptors (mGluRs) will not be covered in this article and the following reviews are suggested [37, 91-93]. Suffice it to say that much of the pharmacology of mGluRs has been elucidated on the hemisected rat spinal cord p r e p a r a t i o n with pre- and post- synaptic receptor subtypes being pharmacologically identified [92, 94]. SPINAL CORD PHARMACOLOGY
Methods. Two basic techniques have contributed much to the advances in the pharmacology of glutamate receptors. The first was the in vivo spinal cord preparation using the technique of microelectrophoresis to administer compounds into the region of single neurones of cats and rats whilst making extracellular recordings of their action potential discharges [2, 19]. In this way neurones can be excited by a number of excitatory amino acids and test compounds can then be administered locally or systemically to study any specific effects on particular receptors. Compounds may also be administered while recording synaptic responses so that receptors mediating neurotransmission can be examined. The second technique is the hemisected spinal cord in vitro, initially of amphibia but now more commonly of neonatal rats [6]. Grease-seals or suction electrodes are used to record DC potentials or synaptic responses from ventral or dorsal roots. Since compounds are usually added to the bathing solution, more quantitative information from this in vitro preparation can be obtained than from microelectrophoresis experiments in vivo.
247 Much of the basic pharmacology of glutamate receptors was elucidated from these two spinal cord preparations in vivo or in vitro. Thus, the differential sensitivity of neurones to kainate, quisqualate and NMDA [3], the differential antagonistic effects of DAA, HA-966, GAMS, GDEE, D-AP5, Mg 2+ and ketamine [6-9, 19] and the effects of these antagonists on synaptic transmision [11-13, 95] were initially described in these spinal preparations.
Results. Competitive non-NMDA antagonists. NBQX reduces responses of rat spinal neurones to electrophoretically administered AMPA and kainate. There is no differential effect on these two agonists but responses to NMDA are unaffected [49]. Part of the depolarising response to kainate of the hemisected rat spinal cord in vitro and cortical slices is, however, resistant to NBQX and is interestingly sensitive to the barbiturate, methohexitone [48, 49]. Similarly electrophoretically administered LY293558 [50] and AMOA [53] reduce AMPA and kainate responses in parallel on spinal neurones in vivo whereas, on cortical slices, these two antagonists preferentially reduce responses to AMPA. LY293558 is also active following systemic administration; thus 2, 5 and 10 mg/kg i.v. produced 18+2%, 49+4% and 86+7% inhibitions respectively of responses of spinal neurones to AMPA. The 10mg/kg close reduced NMDA responses by only 8+4%. C h a n n e l blockers. Philanthotoxin, from the Egyptian digger wasp, selectively and potently blocks responses of spinal neurones in vivo to AMPA and kainate but not to NMDA [60]. This observation was extended to argiotoxin 636, Joro spider toxin and Nephila spider toxin [60]. With the exception of argiotoxin, all the toxins showed good selectivity for non-NMDA receptors relative to those for NMDA, but again did not differentiate between AMPA and kainate. The onset of block and recovery was considerably slower than that of electrophoretically administered competitive antagonists and the recovery was clearly dependent on frequency of agonist application suggesting that, as in other preparations, these act as open channel blockers [54, 57]. Allosteric modulators. The 2,3-benzodiazepine, GYKI 52466, was first shown to block spinal reflexes [61] and subsequently to block responses to AMPA on spinal neurones in vivo [63]. Following electrophoretic ejection of GYKI 52466, responses to kainate were reduced in parallel with those to AMPA whereas those to NMDA were unaffected. The block is clearly non-competitive on cortical slices and presumably will also be on spinal cord tissue when tested in vitro. LY300164 (GYKI 53655) was a more potent AMPA antagonist t h a n GYKI 52466 on spinal neurones but had the same selectivity [96]. For example, following intravenous (5mg/kg) or oral (10mg/kg) administration, LY300164 reduces AMPA responses by 80-100% with only minor effects on responses to NMDA. Cyclothiazide administered elctrophoretically increased response to AMPA
248 and kainate to a similar extent with no effect on those to NMDA [97]. Given intravenously lmg/kg, cyclothiazide enhanced responses of 6 spinal neurones to AMPA by 119 + 28 % and those to NMDA by an insignificant 16 + 6%. Again with cyclothiazide, AMPA responses were enhanced to a greater extent than those of kainate on cortical slices [71]. Concanavilin A has not been tested in vivo but on dorsal root ganglion neurones and dorsal root fibres in vitro responses to kainate are enhanced by this plant lectin but not by cyclothiazide [64, 68, 86, 87]. Concanavilin A also enhances responses of low doses of kainate on cortical wedges to a greater extent t h a n those of AMPA (D. Lodge - unpublished observations). Interestingly when tested in the presence of either NBQX or LY300164, cyclothiazide reversed the effects of LY300164 but not those of NBQX [97]. Thus cylothiazide changed the reduction of responses to AMPA by NBQX from 69+5% to 50+6% whereas those by LY300164 were changed from 68+5% to 21+7%. An i n t e r a c t i o n , therefore, seems likely b e t w e e n the cyclothiazide and 2,3benzodiazepine sites on the AMPA receptor complex. DISCUSSION.
On spinal as on other neurones there is a clear separation between nonNMDA and NMDA receptors. Responses to AMPA and kainate are, however, not easily separated pharmacologically in vivo by competitive (NBQX, LY293558 & AMOA) and non-competitive (GYKI 52466, LY300164 & polyamine toxins) antagonists nor by cyclothiazide, a blocker of desensitisation. Hence it appears t h a t AMPA and kainate excite spinal neurones via the same or similar receptors. The effects of 2,3-benzodiazepines and cyclothiazide make it likely that this is an AMPA receptor of the GluR1-4 type, since cyclothiazide and 2,3-benzodiazepines have little effect on the kainate (GluR5-6) subtype of receptors [64, 65, 68, 8587]. F u r t h e r m o r e the activity of Joro spider and other polyamine toxins suggest t h a t the AMPA receptors contain only a low proportion of GluR2 subunits, since receptors including this subunit are insensitive to these toxins [88-90]. Because the effectiveness of Joro spider toxin is dependent on the presence of glutamine r a t h e r t h a n arginine at the Q ~ site on TM2, another possibility is that GluR2 s u b u n i t s are not fully edited in the r a t spinal cord. Both of these two possibilities suggests t h a t AMPA receptors on these spinal neurones are permeable to calcium and hence could mediate calcium induced cell death when glutamate levels are increased as in spinal ischaemia or trauma. The reversal of 2,3-benzodiazepine, b u t not NBQX, a n t a g o n i s m by cyclothiazide is interesting. Initially this might suggest an interaction between the binding sites of these two compounds but it is strange that the response of AMPA during the NBQX administration is not enhanced. In experiments in cortical slices, a similar phenomenon is seen with cyclothiazide shifting the doseresponse curve of 2,3-benzodiazepines, but not of NBQX, to the right but in a non-parallel fashion. In patch clamp experiments on hippocampal neurones the interaction between cyclothiazide and 2,3-benzodiazepines has been suggested to be competitive [98] but on AMPA-evoked noradrenaline release cyclothiazide did
249 not change the IC50 either of these non-competitive or of competitive AMPA antagonists [99]. With such divergent data more experimentation is required. There are two obvious outstanding questions which require attention. Firstly, what is the role of kainate receptor subunits in the spinal cord? These have been demonstrated in localisation experiments. There is high level of GluR5 expression in the dorsal root ganglion [85] and of this and other kainate subunits in the cord proper [100]. On dorsal root ganglia and C fibres, kainate, rather than AMPA, produces depolarisations with desensitising response which was reversed by concanavilin A [64, 68, 86, 87]. This may then represent a pure population of kainate receptors? In the present in vivo experiments, however, no responses to kainate insensitive to the AMPA receptor selective compounds described above were observed, suggesting that there are few true kainate receptors on or near the neuronal soma from which recordings were made. It seems likely that kainate receptors therefore have a dendritic, or more likely a presynaptic, location. In this position they presumably act as autoreceptors. There is supportive evidence of this in the literature both from electrophysiological studies on dorsal root fibres and from release experiments [see 101 & 102]. The NBQX resistant component in grease seal preparations of the neocortex [101] and spinal cord [102] are compatible with this, since depolarisation of terminals would contribute to the signal seen in such experiments. S e c o n d l y , it should be remembered that the original separation of AMPA and kainate receptors came from spinal cord experiments using GDEE and GAMS. Why did these two rather weak antagonists separate between AMPA and kainate responses when the newer, more potent and selective AMPA antagonists do not? One possibility would be that GDEE and GAMS change the receptor configuration into kainate- and AMPA- preferring states respectively which might be equivalent to a desensitised and non-desensitised state. The similarity of the effects of cyclothiazide on response to AMPA and kainate make this seem unlikely. Unfortunately there has been no extensive study of GDEE and GAMS on recombinant receptors which might shed some light on the conundrum.
Acknowledgements:
We would like to thank those Drs Paul Ornstein, Tage Honore and Istvan Tarnawa for supplies of LY293558, NBQX, and GYKI 52466 respectively, and our earlier colleagues Martyn Jones, Sophie Zeman and Andrew Palmer for allowing us to represent their published data.
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Honore T, Davies SN, Drejer J, Fletcher EJ, et al. Science 1988; 241:701703. Sheardown MJ, Nielsen EO, Hansen AJ, Jacobsen P, Honore T. Science 1990; 247:571-574. Lodge D, Jones MG, Palmer AJ. Can J Physiol Pharmacol 1991; 69:1123. Ornstein PL, Arnold MB, Augenstein NK, Lodge D, et al. J Med Chem 1993; 36:2046-2048. Ornstein PL, Schoepp DD, Arnold MB, Augenstein NK, et al. J Med Chem 1992; 35:3547-3560. Ornstein PL, Arnold MB, Augenstein NK, Leander JD, et al. J Med Chem in press. Krogsgaard-Larsen P, Ferkany JW, Nielsen EO, Madsen U, et al. J Med Chem 1991; 34:123-130 Jackson H, Usherwood PNR. Trends Neurosci 1988; 11:276-283. Eldefrawi A, Eldefrawi T, Konno ME, Mansour NA, et al. Proc Natl Acad Sci 1988; 85:4010-4013. Parks TN, Mueller AL, Artman LD, Albensi BC, et al. J Biol Chem 1991; 266:21523-215529. Priestley T, Woodruff GN, Kemp JA. Br J Pharmacol 1989; 97:1315-1323. Ashe JH, Cox CL, Adams ME. Brain Res 1989; 480:234-241. Saito M, Kawai N, Miwa A, Pan-Hou H, Yoshioka M. Brain Res 1985; 481:16-24. Jones MG, Lodge D. Eur J Pharmacol 1991; 204:203-209. Tarnawa I, Farkas S, Bersenyi P, Pataki A, Andrasi, F. Eur J Pharmacol 1990; 167:193-199. Ouardouz M, Durand J. Neurosci Lett 1991; 125:5-8. Lodge D, Jones MG, Palmer AJ, Zeman S. In: Drug Research related to Neuroactive Amino Acids, Schousboe, A., Diemer, N.H. and Kofod, H. (eds,) Munksgaard, Copenhagen. 1992; 153-160. Wilding TJ, Huettner JE. Mol Pharmacol 1995; 47:582-587. Paternain AV, Morales M, Lerma J. Neuron 1995; 14:185-189. Zorumski CF, Thio LL, Clark DB Neuron 1990; 5:61-66. O'Dell TJ, Christensen BN. J Neurophysiol 1989; 61:1097-1109. Huettner JE. Neuron 1990; 5:255-266. Yamada KA, Rothman SM. J Physiol 1992; 458:409-423. Patneau DK, Vyklicky L Jr, Mayer ML. J Neurosci 1993; 13:3496-3509. Palmer AJ, Lodge D. Eur J Pharmacol-Mol Pharmacol 1993; 244:193-194. Hollmann M, O'Shea-Greenfield A, Rogers S, Heinemann S. Nature 1989; 343:643-648. Hollman M, Hartley M, Heinemann S. Science 1991; 252:851-853. Dani JA, Mayer ML. Curr Opin Neurobiol 1995; 5:310-317. Bettler B, Mulle C. Neuropharmacology 1994; 24:123-139. Sommer B, Keinanen K, Verdoorn TA, Wisden W, et al. Science 1990; 249:1580-1585. Wo ZG, Oswald RE. Trends Neurosci 1995; 18: 161-168. Hollmann M, Maron C, Heinemann S. Neuron 1994; 13:1331-1343. Bennett JA, Dingledine R. Neuron 1995; 14:373-384. Stern-Bach Y, Bettler B, Hartley M, Sheppard PO et al. Neuron 1994; 13:1345-1357.
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241
Spinal glutamate receptors David Lodge and Ann Bond Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom
INTRODUCTION Glutamate, and possibly other acidic amino acids, are now accepted as the major mediators of excitatory neurotransmission in the brain and spinal cord. The initial discovery of glutamate's stimulatory effects on neurones of the central nervous system (CNS) stems from work in the 1950s 1,2]. Despite its ubiquitous function in general cellular metabolism, the transmitter role of glutamate was slowly accepted during the 1960s and 70s. This was aided by separate but linked observations in pioneering laboratories. 1. Neurones in different parts of the brain and spinal cord showed different sensitivities to derivatives of glutamate [3,4]. These included synthetic derivatives such as N-methyl-D-aspartate (NMDA) and natural products such as quisqualate and kainate [5]. 2. Secondly, other synthetic derivatives were found to have antagonistic actions against some of the above glutamate analogues and against synaptic excitations in the CNS [6-9]. These included 1-hydroxy-3-aminopyrrolidone-2 (HA-966), D-a-amino-adipate, ~/-glutamyl-amino-methyl-sulphonate (GAMS) and glutamate diethyl ester (GDEE). The former two selectively reduced responses to NMDA, a pharmacological feature which was shared with several divalent cations, such as magnesium [10]. GAMS and GDEE were shown to have selective effects versus quisqualate and kainate induced responses respectively. Additionally, short latency monosynaptic responses were reduced by the quisqualate antagonists whereas longer latency polysynaptic responses were reduced by NMDA antagonists both in vitro and in vivo [11-13]. 3. Neurochemical studies using radiolabelled glutamate and some of the above analogues showed specific binding sites and uptake mechanisms [3, 14, 15]. The presence of these presumed postsynaptic receptors and transporter systems provided key support for the electrophysiological evidence for the transmitter role of L-glutamate. The selectivity of many of these early agonists and antagonists was poor but nevertheless a concept of three glutamate receptor subtypes developed [7, 8, 11, 16, 17], and despite close examination over the years, this subdivision still holds generally true today (see below). NMDA receptors. The NMDA receptor has received considerable attention, largely because of the early development of selective tools for its study. NMDA itself is a specific ligand for this receptor with little action at other types of glutamate receptor. In particular, the discovery of a highly selective and competitive NMDA antagonist
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in D-2-amino-5-phosphonopentanoate (AP5) [17, 18] allowed many aspects of the physiological and pathological role of this receptor type to be elucidated. The discovery that arylcyclohexylamines such as ketamine and phencyclidine and benzomorphans such as cyclazocine and N-allyl-normetazocine (SKF10,047) were non-competitive channel-blocking NMDA antagonists, was particularly helpful for whole animal electrophysiological and behavioural studies, since these compounds, unlike the competitive antagonists, crossed the blood-brain barrier quickly [19,20]. Three other key discoveries provided insights into the physiology of the NMDA receptor. Firstly, the block of the NMDA receptor by magnesium [10] was shown to provide the key voltage-dependent property of this receptor [21]. At resting membrane potentials around -70 to -60 mV, normal extracellular levels of magnesium provide a brake on the permeability of the receptor, but as neurones depolarise due to synaptic inputs (or to metabolic challenges) the magnesium braking action is relieved and the channels conduct current freely. Secondly, the NMDA receptor was shown to be highly permeable to calcium [22]. The resulting increase in intracellular calcium is important not only for physiological, but also for pathological, facilitation of calcium dependent enzymic functions. Thirdly, allosteric modulation of NMDA receptors by glycine acting at a separate recognition domain on the NMDA receptor complex [23]. It is now known that the NMDA receptor requires co-activation of both the glutamate and glycine recognition sites and indeed a co-transmitter role for glycine has been proposed. In the presence of low amounts of glycine the NMDA receptor appears to undergo desensitisation which can be reversed by increasing the external concentration of glycine [24, 25]. It is still a matter of some debate as to whether extracellular levels of glycine are sufficient to fully saturate this site. Nevertheless because of the requirement for co-activation of the glycine site, considerable pharmacological effort has been made to produce partial agonists and full anatgonists at this receptor [25]. Alongside these largely electrophysiological studies, others showed that NMDA receptors mediated profound neurotoxicity [26-30] and have a central role in epileptiform discharges [31-32] in vitro and in vivo. As a result, the therapeutic potential of NMDA receptor antagonists has emerged and several agents are under development by pharmaceutical companies [33]. The first NMDA receptor subunit (now known as NMDA-R1) was cloned and expressed as a functional NMDA receptor channel complex by Moriyoshi, Nakanishi and colleagues [34]. By homology cloning, NMDA-R2 subunits, A,B,C and D, were also identified. NMDA-R2 subunits, unlike NMDA-R1, do not, however, form functional homomeric channels. It has subsequently been shown that native NMDA receptors consist of heteromeric complexes of NMDAR1 associated with NMDA-R2A-D, presumably in a pentameric structure. All such known NMDA receptor configurations have pharmacology in general agreement with that described above, although variations in kinetics and pharmacology have been reported. Constructs with NMDA-R2C are relatively insensitivite to glycine and to Mg 2+, which may correlate with the pharmacology of NMDA receptors of the cerebellum where 2C subunits are highly expressed. As NMDA receptors are not the major subject of this chapter, readers are referred to recent reviews of their molecular biology and pharmacology [13, 3537].
243 AMPA and kainate receptors. Until recently there has been a lack of pharmacological tools for other glutamate receptors. The original subdivision of non-NMDA receptors depended on the agonist selectivity of quisqualate, which has subsequently been shown also activate phospholipase-coupled metabotropic receptors [38]. The synthesis of a new and more selective ligand was therefore a crucial step. The discovery of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) as a GDEE sensitive agonist [39], led to the re-naming of the non-NMDA receptors as AMPA and kainate receptors [16, 40]. Furthermore radioligand binding experiments have allowed separate high affinity [3H]AMPA and [3H] kainate binding sites to be distinguished [14, 40, 41]. Some cross displacement, particularly of [3H]AMPA by unlabelled kainate, however, confirmed electrophysiological observations of some non-selectivity of these two agonists, which still remains a problem. The characteristic pharmacology of AMPA was subsequently corellated, following the use of patch clamp techniques, with its kinetic profile; thus, AMPA evoked rapidly desensitising responses in central neurones whereas those to kainate were relatively non-desensitising [42, 43]. Block of AMPA currents, however, by prior application of kainate suggested a common site but with different mechanisms, AMPA acting as a partial agonist and kainate as a full agonist [42, 44-46]. On this basis, the separation between kainate and AMPA receptors was unlikely to be achieved without other selective agents or molecular biological identification of distinct receptor subunits. Both these advances have been made in the last five years.
Competitive AMPA receptor antagonists. The low margin of selectivity between AMPA and kainate responses with the early antagonists such as GDEE and GAMS especially in in vitro studies required the development of more potent and selective competitive antagonists. The first such compounds showing selective AMPA antagonist activity were the 6-cyano-7-nitro- and 6,7-dinitro-quinoxalinediones, CNQX and DNQX, [47] but their selectivity for AMPA over kainate was not more than five-fold. A further synthetic development led to 3-dihydroxy-6-nitro-7-sulphamoyl-benz(F)quinoxaline (NBQX) which has a 30-fold greater selectivity at displacing the binding of [3H]AMPA, rather than that of [3H]kainate [48]. NBQX also has similar 30 fold higher potency as an antagonist of AMPA-evoked depolarisations on cortical slices in vitro [48, 49]. In a later development of competitive AMPA receptor antagonists, LY293558, a decahydroisoquinoline with a tetrazole group substituted at the 6 position, was shown to selectively displace [3H]AMPA binding on rat brain membranes and to antagonise AMPA-induced depolarisations on cortical slices and, following systemic administration, to block central effects of AMPA receptor agonists [50]. This compound is also interesting from a medicinal chemistry stand-point. In LY293558 the decahydroisoquinoline is separated from the tetrazole by 2 carbons [50]; a single carbon in this chain yields a selective NMDA antagonist, LY233536 [51] whereas a direct carbon linker gives a non-selective NMDA and AMPA antagonist, LY246492 [52].
244 Similar data to that with LY293558 has been reported for two analogues of AMPA called AMOA and AMNH which respectively are substituted with a 3carboxymethoxy and a 2-methylisoxazole on the isoxazole ring of AMPA [53]. AMOA selectively displaces [3H]AMPA binding whereas AMOA displaced [3H]AMPA and low affinity [3H]kainate binding more or less equally; neither compound displaced high affinity [3H]kainate binding. On cortical slices AMOA and AMNH were weakly selective for AMPA- and kainate- induced depolarisations respectively [53]. AMPA r e c e p t o r channel blockers A number of invertebrate polyamine toxins have recently been shown to be open channel blockers of glutamate receptors initially at the neuromuscular junction of locusts [54] and recently on central mammalian neurones [54-57]. These toxins include argiotoxin 636, Joro spider toxin and philanthotoxin. Although these polyamine toxins block NMDA receptors at least in some preparations, in others they also block responses mediated by AMPA receptors more selectively [58-60].
Allosteric modulators of AMPA receptors. In addition to the above competitive antagonists, a 2,3-benzodiazepine, GYKI 52466 [61-63] has highlighted a new mechanism of modulating non-NMDA receptors in a non-competitive manner. GYKI 52466, selectively reduces responses to AMPA and not to NMDA, and is about one fifth as potent against kainate responses, on rat cortical slices [63]. This is, therefore, another useful tool for separating between AMPA and kainate receptors. LY300164 (GYKI 53655), the 3-methyl-carbamoyl derivative of GYKI 52466, is approximately 10 times more potent than GYKI 52466 on AMPA receptors but less potent on kainate receptors [64, 65]. As desensitisation is a prominent feature of AMPA receptors, a plant lectin, concanavilin A, previously shown to block desensitisation of insect glutamate receptors [54], was tested and found to enhance AMPA and kainate responses on hippocampal [43, 66], retinal [67] and dorsal root ganglion (DRG) [68] neurones. More recently, a series of benzothiazides including diazoxide and cyclothiazide, were shown to reduce glutamate receptor desensitisation and hence increase AMPA responses on hippocampal [69, 70] and cortical [71] neurones. As will be discussed below, it has become clear that concanavilin A and cyclothiazide act selectively on rapidly inactivating kainate and AMPA responses respectively. Molecular biology of AMPA and kainate receptors. An AMPA receptor, GluR1 (or GluRA) was the first glutamate receptor to be cloned and expressed [72]. Our concepts of AMPA and kainate receptors have been revolutionised over the past six years through the application of molecular biology techniques to this area. In the ensuing five years, the total number of non-NMDA receptor subunits has increased to nine. They can be divided into two major types: a) the AMPA receptors, GluR1-4, which bind AMPA with high affinity and have a rapidly desensitising response to this agonist and a smaller but non-desensitising response to kainate. These channels with the exception of GluR2 are permeable to calcium [73]. In the proposed TM1 region of GluR2 an
245 arginine occurs at the site at which glutamine is positioned in GluR1,2 and 4. This so called Q/R site has been shown by site directed mutagenesis studies to be a major determinant of calcium permeability [35-37]. b) the kainate receptors, GluR5-7 and KA1-2 which bind kainate with medium and high affinity respectively [35-37] Only GluR5-6 form homomeric channels whereas the others require heteromeric expression with GluR5 or 6 to become functional. GluR5 and 6 also have the Q/R site in the TM2 region and in this case editing appears to be variable throughout the CNS [35-37]. Only a small part of the molecular biology will be covered here; more comprehensive reviews are recommended [35-37, 74, 75]. Although the molecular pharmacology of glutamate receptors is still in its infancy, some interesting data are emerging:1. The calcium permeability of non-NMDA receptors is controlled largely by the so called Q/R site in the putative second transmembrane segment (TM2). This site is occupied by glutamine (Q) in GluRsl, 3 and 4 but by an arginine (R) in GIuR2. This arginine accounts for the low permeability to calcium of both homomeric or hetromeric channels with GluR2. The equivalent site in the NMDA receptors is occupied by an asparagine which may account for the calcium permeability and magnesium block of the NMDA channel. The genomic DNA codes for a glutamine at this Q/R site even in GluR2; so editing of RNA (CAG to CIG by adenosine deaminase) results in an arginine in the GluR2 protein. 2. Desensitisation is controlled in part by alternative splicing of the so-called flip and flop region in the loop between TM3 and TM4 [76]. The presence of glycosylation sites [77-79] and at least part of the agonist binding domain [80] in the same loop suggests its extracellular location. 3. The topology of the glutamate receptor as a result of such studies with m u t a n t receptors is now thought to be quite different from the four transmembrane crossing model suggested for nicotinic receptors, etc. [74,77-79]. TM2 is thought to loop within the membrane rather than crossing it, which in turn leads to an extracellular TM3-4 loop and an intracellular carboxy terminus. The Q/R site is thought to be near the apex of the intramembrane TM2 loop. This model, which has similarities with voltage-dependent ion channels, highlights the limitations of interpretations based solely on hydrophobicity plots. 4. NBQX and CNQX are relatively non-selective antagonists across all the recombinant non-NMDA receptors. Thus NBQX blocks GluR1-4 receptors in the 100nM region [81, 82] and GluR5 and 6 in the 2mM region [83, 84]. 5. By contrast LY293558, tested on human recombinant receptors, displaces GluR1-4 binding with 2-40uM potency, and 900nM potency on GluR5 but is almost inactive (mM) on GluR6 (R.J. Kamboj - unpublished observations). This selectivity is also seen in patch clamp studies [84]. Thus LY293558 could be used to differentiate between GluR5 and GluR6 based kainate receptors, although the situation in GluR6 heteromerics is unkown. 6. GYKI 52466, and other more potent 2,3-benzodiazepines, are active on GluR1-4 with only small effects on the kainate preferring subtypes. Thus, LY300164 (GYKI 53655) is effective at lmM as an antagonist of recombinant GluR1-4 and native AMPA receptors whereas 100uM is required to block native and recombinant kainate receptors (64,65, D. B l e a k m a n unpublished observations).
246 7. Interestingly the two blockers of desensitisation, cyclothiazide and concanavilin A, show complimetary selectivities. Cyclothiazide blocks the desensitisation on recombinant GluR1-4 but, if anything, reduces responses on on kainate receptors, GluR5 and 6 [85]. Concanavilin A on the other hand markedly potentiates responses on these kainate receptors but is only weakly effective on the recombinant AMPA receptors [85]. Such results parallel those from the GluR1-4-dominated hippocampal neurones and the GluR5-dominated dorsal root ganglion cells [64, 68, 86, 87] and show the usefulness of these compounds in differentiating between AMPA and kainate receptors. 8. Argiotoxin 636, Joro spider toxin and philanthotoxin blocks AMPA receptors GluR1, 3 and 4 but is much less active on the homomeric or heteromeric channels containing GluR2 and GluR6 [88-90]. GluR6 may also be edited to an arginine at the Q ~ site. Hence this differential effect of toxins has been related to the Q/R site editing of GluR2 and unedited, glutaminecontaining, versions of GluR2 and GluR6 are sensitive to Joro spider toxin. Similarly conversion to an arginine at the Q/R site of GluR4 renders it insensitive to argiotoxin. So the two properties of calcium permeability and toxin efficacy appear to be controlled at least partially by the molecular entities in the channel.
Metabotropic glutamate receptors.
In addition to ion channel coupled receptors, glutamate also activates second messenger systems via G-protein coupled receptors. The molecular biology and pharmacology of metabotropic glutamate receptors (mGluRs) will not be covered in this article and the following reviews are suggested [37, 91-93]. Suffice it to say that much of the pharmacology of mGluRs has been elucidated on the hemisected rat spinal cord p r e p a r a t i o n with pre- and post- synaptic receptor subtypes being pharmacologically identified [92, 94]. SPINAL CORD PHARMACOLOGY
Methods. Two basic techniques have contributed much to the advances in the pharmacology of glutamate receptors. The first was the in vivo spinal cord preparation using the technique of microelectrophoresis to administer compounds into the region of single neurones of cats and rats whilst making extracellular recordings of their action potential discharges [2, 19]. In this way neurones can be excited by a number of excitatory amino acids and test compounds can then be administered locally or systemically to study any specific effects on particular receptors. Compounds may also be administered while recording synaptic responses so that receptors mediating neurotransmission can be examined. The second technique is the hemisected spinal cord in vitro, initially of amphibia but now more commonly of neonatal rats [6]. Grease-seals or suction electrodes are used to record DC potentials or synaptic responses from ventral or dorsal roots. Since compounds are usually added to the bathing solution, more quantitative information from this in vitro preparation can be obtained than from microelectrophoresis experiments in vivo.
247 Much of the basic pharmacology of glutamate receptors was elucidated from these two spinal cord preparations in vivo or in vitro. Thus, the differential sensitivity of neurones to kainate, quisqualate and NMDA [3], the differential antagonistic effects of DAA, HA-966, GAMS, GDEE, D-AP5, Mg 2+ and ketamine [6-9, 19] and the effects of these antagonists on synaptic transmision [11-13, 95] were initially described in these spinal preparations.
Results. Competitive non-NMDA antagonists. NBQX reduces responses of rat spinal neurones to electrophoretically administered AMPA and kainate. There is no differential effect on these two agonists but responses to NMDA are unaffected [49]. Part of the depolarising response to kainate of the hemisected rat spinal cord in vitro and cortical slices is, however, resistant to NBQX and is interestingly sensitive to the barbiturate, methohexitone [48, 49]. Similarly electrophoretically administered LY293558 [50] and AMOA [53] reduce AMPA and kainate responses in parallel on spinal neurones in vivo whereas, on cortical slices, these two antagonists preferentially reduce responses to AMPA. LY293558 is also active following systemic administration; thus 2, 5 and 10 mg/kg i.v. produced 18+2%, 49+4% and 86+7% inhibitions respectively of responses of spinal neurones to AMPA. The 10mg/kg close reduced NMDA responses by only 8+4%. C h a n n e l blockers. Philanthotoxin, from the Egyptian digger wasp, selectively and potently blocks responses of spinal neurones in vivo to AMPA and kainate but not to NMDA [60]. This observation was extended to argiotoxin 636, Joro spider toxin and Nephila spider toxin [60]. With the exception of argiotoxin, all the toxins showed good selectivity for non-NMDA receptors relative to those for NMDA, but again did not differentiate between AMPA and kainate. The onset of block and recovery was considerably slower than that of electrophoretically administered competitive antagonists and the recovery was clearly dependent on frequency of agonist application suggesting that, as in other preparations, these act as open channel blockers [54, 57]. Allosteric modulators. The 2,3-benzodiazepine, GYKI 52466, was first shown to block spinal reflexes [61] and subsequently to block responses to AMPA on spinal neurones in vivo [63]. Following electrophoretic ejection of GYKI 52466, responses to kainate were reduced in parallel with those to AMPA whereas those to NMDA were unaffected. The block is clearly non-competitive on cortical slices and presumably will also be on spinal cord tissue when tested in vitro. LY300164 (GYKI 53655) was a more potent AMPA antagonist t h a n GYKI 52466 on spinal neurones but had the same selectivity [96]. For example, following intravenous (5mg/kg) or oral (10mg/kg) administration, LY300164 reduces AMPA responses by 80-100% with only minor effects on responses to NMDA. Cyclothiazide administered elctrophoretically increased response to AMPA
248 and kainate to a similar extent with no effect on those to NMDA [97]. Given intravenously lmg/kg, cyclothiazide enhanced responses of 6 spinal neurones to AMPA by 119 + 28 % and those to NMDA by an insignificant 16 + 6%. Again with cyclothiazide, AMPA responses were enhanced to a greater extent than those of kainate on cortical slices [71]. Concanavilin A has not been tested in vivo but on dorsal root ganglion neurones and dorsal root fibres in vitro responses to kainate are enhanced by this plant lectin but not by cyclothiazide [64, 68, 86, 87]. Concanavilin A also enhances responses of low doses of kainate on cortical wedges to a greater extent t h a n those of AMPA (D. Lodge - unpublished observations). Interestingly when tested in the presence of either NBQX or LY300164, cyclothiazide reversed the effects of LY300164 but not those of NBQX [97]. Thus cylothiazide changed the reduction of responses to AMPA by NBQX from 69+5% to 50+6% whereas those by LY300164 were changed from 68+5% to 21+7%. An i n t e r a c t i o n , therefore, seems likely b e t w e e n the cyclothiazide and 2,3benzodiazepine sites on the AMPA receptor complex. DISCUSSION.
On spinal as on other neurones there is a clear separation between nonNMDA and NMDA receptors. Responses to AMPA and kainate are, however, not easily separated pharmacologically in vivo by competitive (NBQX, LY293558 & AMOA) and non-competitive (GYKI 52466, LY300164 & polyamine toxins) antagonists nor by cyclothiazide, a blocker of desensitisation. Hence it appears t h a t AMPA and kainate excite spinal neurones via the same or similar receptors. The effects of 2,3-benzodiazepines and cyclothiazide make it likely that this is an AMPA receptor of the GluR1-4 type, since cyclothiazide and 2,3-benzodiazepines have little effect on the kainate (GluR5-6) subtype of receptors [64, 65, 68, 8587]. F u r t h e r m o r e the activity of Joro spider and other polyamine toxins suggest t h a t the AMPA receptors contain only a low proportion of GluR2 subunits, since receptors including this subunit are insensitive to these toxins [88-90]. Because the effectiveness of Joro spider toxin is dependent on the presence of glutamine r a t h e r t h a n arginine at the Q ~ site on TM2, another possibility is that GluR2 s u b u n i t s are not fully edited in the r a t spinal cord. Both of these two possibilities suggests t h a t AMPA receptors on these spinal neurones are permeable to calcium and hence could mediate calcium induced cell death when glutamate levels are increased as in spinal ischaemia or trauma. The reversal of 2,3-benzodiazepine, b u t not NBQX, a n t a g o n i s m by cyclothiazide is interesting. Initially this might suggest an interaction between the binding sites of these two compounds but it is strange that the response of AMPA during the NBQX administration is not enhanced. In experiments in cortical slices, a similar phenomenon is seen with cyclothiazide shifting the doseresponse curve of 2,3-benzodiazepines, but not of NBQX, to the right but in a non-parallel fashion. In patch clamp experiments on hippocampal neurones the interaction between cyclothiazide and 2,3-benzodiazepines has been suggested to be competitive [98] but on AMPA-evoked noradrenaline release cyclothiazide did
249 not change the IC50 either of these non-competitive or of competitive AMPA antagonists [99]. With such divergent data more experimentation is required. There are two obvious outstanding questions which require attention. Firstly, what is the role of kainate receptor subunits in the spinal cord? These have been demonstrated in localisation experiments. There is high level of GluR5 expression in the dorsal root ganglion [85] and of this and other kainate subunits in the cord proper [100]. On dorsal root ganglia and C fibres, kainate, rather than AMPA, produces depolarisations with desensitising response which was reversed by concanavilin A [64, 68, 86, 87]. This may then represent a pure population of kainate receptors? In the present in vivo experiments, however, no responses to kainate insensitive to the AMPA receptor selective compounds described above were observed, suggesting that there are few true kainate receptors on or near the neuronal soma from which recordings were made. It seems likely that kainate receptors therefore have a dendritic, or more likely a presynaptic, location. In this position they presumably act as autoreceptors. There is supportive evidence of this in the literature both from electrophysiological studies on dorsal root fibres and from release experiments [see 101 & 102]. The NBQX resistant component in grease seal preparations of the neocortex [101] and spinal cord [102] are compatible with this, since depolarisation of terminals would contribute to the signal seen in such experiments. S e c o n d l y , it should be remembered that the original separation of AMPA and kainate receptors came from spinal cord experiments using GDEE and GAMS. Why did these two rather weak antagonists separate between AMPA and kainate responses when the newer, more potent and selective AMPA antagonists do not? One possibility would be that GDEE and GAMS change the receptor configuration into kainate- and AMPA- preferring states respectively which might be equivalent to a desensitised and non-desensitised state. The similarity of the effects of cyclothiazide on response to AMPA and kainate make this seem unlikely. Unfortunately there has been no extensive study of GDEE and GAMS on recombinant receptors which might shed some light on the conundrum.
Acknowledgements:
We would like to thank those Drs Paul Ornstein, Tage Honore and Istvan Tarnawa for supplies of LY293558, NBQX, and GYKI 52466 respectively, and our earlier colleagues Martyn Jones, Sophie Zeman and Andrew Palmer for allowing us to represent their published data.
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