Jeffrey C. Watkins

THE EFFECTS OF A SERIES OF ω‐PHOSPHONIC α‐CARBOXYLIC AMINO ACIDS ON ELECTRICALLY EVOKED AND EXCITANT AMINO ACID‐INDUCED RESPONSES IN ISOLATED SPINAL CORD PREPARATIONS

1 The depressant actions on evoked electrical activity and the excitant amino acid antagonist properties of a range of ω‐phosphonic α‐carboxylic amino acids have been investigated in the isolated spinal cord preparations of the frog or immature rat. 2 When tested on dorsal root‐evoked ventral root potentials, members of the homologous series from 2‐amino‐5‐phosphonovaleric acid to 2‐amino‐8‐phosphonooctanoic acid showed depressant actions which correlated with the ability of the substances to antagonize selectively motoneuronal depolarizations induced by N‐methyl‐d‐aspartate. 3 2‐Amino‐5‐phosphonovalerate was the most potent substance of the series giving an apparent KD of 1.4 μm for the antagonism of responses to N‐methyl‐d‐aspartate. 4 A comparison of the (+)‐ and (—)‐forms of 2‐amino‐5‐phosphonovalerate indicated that the N‐methyl‐d‐aspartate antagonist activity and the neuronal depressant action of this substance were both due mainly to the (—)‐isomer. 5 The (—)‐ and (+)‐forms of 2‐amino‐4‐phosphonobutyrate had different actions. The (—)‐form of this substance had a relatively weak and non‐selective antagonist action on depolarizations induced by N‐methyl‐d‐aspartate, quisqualate and kainate and a similarly weak depressant effect when tested on evoked electrical activity. The (+)‐form was more potent than the (—)‐form in depressing electrically evoked activity but did not antagonize responses to amino acid excitants. At concentrations higher than those required to depress electrically evoked activity, the (+)‐form produced depolarization. This action was blocked by 2‐amino‐5‐phosphonovalerate.

2-Amino-5-phosphonovalerate (2APV), a potent and selective antagonist of amino acid-induced and synaptic excitation

A new compound, 2-amino-5-phosphonovaleric acid (2APV) is the most potent and selective N-methyl-D-aspartate (NMDA) receptor antagonist yet tested. As with other compounds of this type, it blocks L-aspartate and dorsal root-evoked excitation of spinal neurons, but is without effect on the cholinergic excitation of Renshaw cells evoked by exogenous acetylcholine or ventral root stimulation. The high potency and selectivity of this compound should prove to be of great value in investigations of the amino acid receptor types involved in synaptic excitation.

The NMDA receptor

2) Agonists and competitive antagonists: structure, activity and molecular modelling studies 3) Non-competitive antagonists of N-methyl-D-aspartate 4) Molecular pharmacology of NMDA receptors 5) Molecular biology of NMDA receptors 6) Anatomical, pharmacological, and molecular diversity of native NMDA receptor subtypes 7) The NMDA receptor, its channel, and its modulation by glycine 8) The time-course of NMDA receptor-mediated synaptic currents 9) Activation of NMDA receptors 10) NMDA receptors and their interactions with other excitatory amino acid receptors in synaptic transmission in the mammalian central nervous system 11) The importance of NMDA receptors in the processing of spinal primary afferent input patterns 12 ) The role of NMDA receptors in synaptic integration and the organization of motor patterns 13) NMDA receptors and long-term potentiation in the hippocampus 14) NMDA receptors and developmental plasticity in visual neocortex 15) The role of NMDA receptors in learning and memory 16) Clinical implications of NMDA receptors 17) The NMDA receptor in epilepsy 18) NMDA receptors, neuronal development, and neurodegeneration 19) Competitive NMDA antagonists as drugs 20) Non-competitive NMDA antagonists as drugs 1) The NMDA receptor concept: origins and development

CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for [3H]d-AP5 binding sites on brain membranes and anticonvulsant activity

Properties of a new potent antagonist acting selectively at N-methyl-D-aspartate (NMDA) type excitatory amino acid receptors are described. This compound, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is more potent than all previously reported NMDA antagonists in depressing mammalian spinal neuronal responses (cat and immature rat), in its affinity for [3H]D-AP5 (a radiolabelled NMDA antagonist) binding sites on rat brain membranes, and as an anticonvulsant in mice.

Competitive antagonism at metabotropic glutamate receptors by (S) -4-carboxyphenylglycine and (RS) -α-methyl-4-carboxyphenylglycine

Two phenylglycine derivates, (S)-4-carboxyphenylglycine and (RS)-alpha-methyl-4-carboxyphenylglycine, competitively antagonised (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD)-stimulated phosphoinositide hydrolysis in rat cerebral cortical slices. The same phenylglycine derivatives selectively antagonized ACPD-induced depolarization in neonatal rat spinal motoneurones and rate thalamic neurones relative to depolarization or excitation induced by N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Both phenylglycine derivatives also selectively depressed synaptic excitation in thalamic neurones evoked by noxious thermal stimuli, without affecting the synaptic stimulation of the same cells by non-noxious stimuli.

Actions of two new antagonists showing selectivity for different sub-types of metabotropic glutamate receptor in the neonatal rat spinal cord

1 The presynaptic depressant action of l‐2‐amino‐4‐phosphonobutyrate (l‐AP4) on the monosynaptic excitation of neonatal rat motoneurones has been differentiated from the similar effects produced by (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate ((1S,3R)‐ACPD), (1S,3S)‐ACPD and (2S,3S,4S)‐α‐(carboxycyclopropyl)glycine (l‐CCG‐I), and from the postsynaptic motoneuronal depolarization produced by (1S,3R)‐ACPD, by the actions of two new antagonists, α‐methyl‐l‐AP4 (MAP4) and α‐methyl‐l‐CCG‐I (MCCG). Such selectivity was not seen with a previously reported antagonist, (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG). 2 MAP4 selectively and competitively antagonized the depression of monosynaptic excitation produced by l‐AP4 (KD 22 μm). At ten fold higher concentrations, MAP4 also antagonized synaptic depression produced by l‐CCG‐I but in an apparently non‐competitive manner. MAP4 was virtually without effect on depression produced by (1S,3R)‐ or (1S,3S)‐ACPD. 3 MCCG differentially antagonized the presynaptic depression produced by the range of agonists used. This antagonist had minimal effect on l‐AP4‐induced depression. The antagonism of the synaptic depression effected by (1S,3S)‐ACPD and l‐CCG‐I was apparently competitive in each case but of varying effectiveness, with apparent KD values for the interaction between MCCG and the receptors activated by the two depressants calculated as 103 and 259 μm, respectively. MCCG also antagonized the presynaptic depression produced by (1S,3R)‐ACPD. 4 Neither MAP4 nor MCCG (200–500 μm) significantly affected motoneuronal depolarizations produced by (1S,3R)‐ACPD. At the same concentrations the two antagonists produced only very weak and variable effects (slight antagonism or potentiation) on depolarizations produced by (S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) and N‐methyl‐d‐aspartate (NMDA). 5 It is concluded that MAP4 is a potent and selective antagonist for those excitatory amino acid (EAA) receptors on neonatal rat primary afferent terminals that are preferentially activated by l‐AP4, and that MCCG is a relatively selective antagonist for different presynaptic EAA receptors that are preferentially activated by (1S,3S)‐ACPD and (perhaps less selectively) by l‐CCG‐I. These receptors probably comprise two sub‐types of metabotropic glutamate receptors negatively linked to adenylyl cyclase activity.

Structure-activity relations of excitatory amino acids on frog and rat spinal neurones.

1 A series of compounds structurally related to glutamic acid has been tested on frog and rat spinal neurones. The substances were added to procaine‐containing medium bathing the isolated hemisected spinal cord of the frog, and their potencies in depolarizing motoneurones were assessed by the magnitude of the potential produced in the ventral root. The electrophoretic technique was used to administer the substances around single interneurones of the rat spinal cord and the relative potencies of the compounds as excitants assessed by the magnitude of the currents required to produce similar rates of neuronal firing. 2 Parallel structure‐activity relations were observed in the two series of experiments, suggesting that the receptors for excitatory amino acids on frog and rat spinal neurones are similar. 3 Quisqualate, domoate and kainate were the strongest excitants in both animals, with potencies around two orders of magnitude higher than that of l‐glutamate. 4 2,4,5‐Trihydroxyphenylalanine (6‐OH‐DOPA) was a stronger excitant and l‐3,4‐dihydroxyphenylalanine (l‐DOPA) a weaker excitant than l‐glutamate on frog spinal motoneurones. The former compound was also a more potent convulsant than l‐glutamate on intraventricular injection into mouse brain. The lack of activity of 6‐OH‐DOPA on electrophoretic administration was attributed to oxidation. 5 Unlike the majority of amino acid excitants, several of the compounds shown in the present work to have moderate excitatory activity are not anionic at physiological pH. This indicates either that two negatively charged groups are not essential for interaction with a common excitatory receptor, or that more than one type of receptor is involved in the actions demonstrated.

The glutamate story

Glutamatergic synaptic transmission in the mammalian central nervous system was slowly established over a period of some 20 years, dating from the 1950s. Realisation that glutamate and like amino acids (collectively known as excitatory amino acids (EAA)) mediated their excitatory actions via multiple receptors preceded establishment of these receptors as synaptic transmitter receptors. EAA receptors were initially classified as N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors, the latter subdivided into quisqualate (later AMPA) and kainate receptors after agonists that appeared to activate these receptors preferentially, and by their sensitivity to a range of differentially acting antagonists developed progressively during the 1970s. NMDA receptors were definitively shown to be synaptic receptors on spinal neurones by the sensitivity of certain excitatory pathways in the spinal cord to a range of specific NMDA receptor antagonists. Importantly, specific NMDA receptor antagonists appeared to be less effective at synapses in higher centres. In contrast, antagonists that also blocked non‐NMDA as well as NMDA receptors were almost universally effective at blocking synaptic excitation within the brain and spinal cord, establishing both the existence and ubiquity of non‐NMDA synaptic receptor systems throughout the CNS. In the early 1980s, NMDA receptors were shown to be involved in several central synaptic pathways, acting in concert with non‐NMDA receptors under conditions where a protracted excitatory postsynaptic potential was effected in response to intense stimulation of presynaptic fibres. Such activation of NMDA receptors together with non‐NMDA receptors led to the phenomenon of long‐term potentiation (LTP), associated with lasting changes in synaptic efficacy (synaptic plasticity) and considered to be an important process in memory and learning. During the 1980s, it was shown that certain glutamate receptors in the brain mediated biochemical changes that were not susceptible to NMDA or non‐NMDA receptor antagonists. This dichotomy was resolved in the early 1990s by the techniques of molecular biology, which identified two families of glutamate‐binding receptor proteins (ionotropic (iGlu) and metabotropic (mGlu) receptors). Development of antagonists binding to specific protein subunits is currently enabling precise identification of discrete iGlu or mGlu receptor subtypes that participate in a range of central synaptic processes, including synaptic plasticity.

1S,3R-ACPD stimulates and L-AP3 blocks Ca2+ mobilization in rat cerebellar neurons.

We have used digital fluorescence imaging to compare the ability of the separate enantiomers of trans-ACDP to mobilize intracellular free calcium in cerebellar granule cells, maintained in primary culture. In addition, we have examined the ability of the separate D and L enantiomers of AP3 to antagonise this calcium mobilizing response

Phenylglycine derivatives as new pharmacological tools for investigating the role of metabotropic glutamate receptors in the central nervous system.

The possible roles of G-protein coupled metabotropic glutamate receptors in central nervous function are currently the focus of intensive investigation. The complexity of effects produced by agonists at these receptors probably reflects the activity of a range of sub-types. The metabotropic glutamate receptors first described are linked to phospholipase C, mediating phosphoinositide hydrolysis and release of Ca2+ from intracellular stores. A substance generally considered to be a selective agonist for these receptors is (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). This substance not only stimulates phosphoinositide hydrolysis, but also inhibits cyclic AMP formation. A family of metabotropic glutamate receptors, incorporating both phospholipase C- and adenylcyclase-linked sub-types has been cloned. Various effects of metabotropic glutamate receptor agonists on membrane ion fluxes and synaptic events have been reported, including neuronal depolarization and/or excitation, hyperpolarization, inhibition of Ca(2+)-dependent and voltage-gated K+ currents, potentiation of N-methyl-D-aspartate-induced responses, depression of synaptic excitation and either induction or augmentation of long-term potentiation. To clarify the role of metabotropic glutamate receptors in central nervous activity and to aid the characterization of the various receptor types that may be involved, a range of highly selective agonists and antagonists is required. To date, currently available antagonists such as L-2-amino-3-phosphonopropionate and L-aspartic acid-beta-hydroxamate appear to be unselective and insufficiently potent. We report here the actions of three phenylglycine derivatives, the particular agonist and/or antagonist properties of which may help to elucidate the roles of metabotropic glutamate receptors in central nervous activity.(ABSTRACT TRUNCATED AT 250 WORDS)