D. Lodge

A new class of GABA agonist

COMPOUNDS which mimic the activity of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) on postsynaptic receptors are of particular pharmacological interest as possible therapeutic agents in human neurological disorders and as molecular probes with which to study different types of GABA receptors. The GABA molecule has considerable flexibility with free rotation around all three carbon–carbon bonds as indicated in Fig. 1. This conformational mobility is reduced in GABA analogues such as trans-4-aminocrotonic acid1 and muscimol2, which are potent GABA agonists at bicuculline-sensitive receptors on spinal neurones of the cat. The conformational mobility of such compounds can be reduced still further by incorporating the amino function in a ring structure. This has led to a new class of GABA agonist described here, based on l,2,3,6-tetrahydropyridine-4-carboxylic acid (isoguvacine) and the related bicyclic isoxazole 4,5,6,7-tetra-hydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (Fig. 1). Isoguvacine represents a semi-rigid analogue of trans-4-aminocrotonic acid in a folded conformation. THIP, a folded analogue of muscimol, is even more rigid as the ‘masked’ carboxyl group (the 3-isoxazolo moiety) is fixed in the general plane of the molecule. The action of these compounds as GABA agonists provides indirect evidence that GABA interacts with bicuculline-sensitive postsynaptic receptors in the cat spinal cord in a partially extended and almost planar conformation.

DIHYDROMUSCIMOL, THIOMUSCIMOL AND RELATED HETEROCYCLIC COMPOUNDS AS GABA ANALOGUES

A series of heterocyclic GABA analogues related to muscimol (5‐aminomethyl‐3‐isoxazolol) were tested as depressants of the firing of GABA sensitive neurones on the cat spinal cord, and as inhibitors of the sodium‐independent binding of GABA to rat brain membranes. Furthermore, the compounds were examined as inhibitors of GABA uptake into rat brain slices and as inhibitors of the activities of the GABA‐metabolizing enzymes L‐glutamate 1‐carboxylyase and GABA:2‐oxoglutarate aminotransferase.

Selective effects of (-)-baclofen on spinal synaptic transmission in the cat.

SummaryWhen ejected microelectrophoretically near spinal interneurones of cats anaesthetised with pentobarbitone and under conditions where postsynaptic excitability was maintained artificially at a constant level, (−), but not (+), -baclofen selectively reduced monosynaptic excitation by impulses in low threshold muscle (Ia and Ib) and cutaneous (Aα) afferents. Polysynaptic excitation of interneurones and Renshaw cells by impulses in higher threshold afferents was less affected, and baclofen had little or no effect on the cholinergic monosynaptic excitation of Renshaw cells. Glycinergic and gabergic inhibitions of spinal neurones were relatively insensitive to baclofen. These stereospecific actions of baclofen, produced by either a reduction in the release of excitatory transmitter or postsynaptic antagonism, suggest that Ia, Ib, and Aα afferents may release the same excitatory transmitter which differs from that of spinal excitatory interneurones.Microelectrophoretic (−), but not (+), -baclofen also reduced primary afferent depolarization of ventral horn Ia extensor afferent terminations produced by impulses in low threshold flexor afferents, without altering either the electrical excitability of the terminations or their depolarization by electrophoretic GABA or L-glutamate. This stereospecific action of baclofen is interpreted as a reduction in the release of GABA at depolarizing axo-axonic synapses on Ia terminals.

The depolarization of feline ventral horn group Ia spinal afferent terminations by GABA

SummaryThe unmyelinated terminal regions of extensor muscle Ia afferent fibres were stimulated electrically near lumbar motoneurones in anaesthetised cats using 300 μs pulses of less than 1 μA passed through the central NaCl barrel of seven barrel micropipettes. Such terminations were identified by anodal blocking factors of less than four and the latency of the antidromic impulse recorded in the appropriate peripheral muscle nerve.Although the effects of microelectrophoretically administered GABA were occasionally complex, the most consistent finding was a reduction in termination threshold followed by an increase. Both this reduction in threshold by GABA, and that produced by tetanic stimulation of low threshold flexor afferents (PAD) were diminished by microelectrophoretic bicuculline methochloride. This GABA antagonist alone elevated the threshold of some terminations but did not reduce the depolarizing action of either potassium or L-glutamate. Furthermore, since reductions in threshold by GABA, but not by either potassium or L-glutamate, were associated with a decrease in PAD, GABA appears to increase terminal membrane conductance.Since neither GABA nor bicuculline methochloride influenced the threshold or afferent depolarization of non-terminal regions of Ia fibres, these results are consistent with the function of GABA as a depolarizing transmitter at gabergic axoaxonic synapses upon the terminals of Ia afferent fibres synapsing with motoneurones.

Muscimol uptake, release and binding in rat brain slices.

Abstract— The GABA analogue, muscimol, was taken up relatively inefficiently compared to GABA by slices of rat cerebral cortex at 37 C. Muscimol uptake followed saturation kinetics (Km ImM. Vm 0.1 μmol g mini and showed an absolute dependence on sodium ions. The relative susceptibilities of muscimol uptake and GABA high affinity uptake to a variety of inhibitors, including (‐)‐nipecotic acid. (+)‐2.4‐diaminobutyric acid and arecaidine, and the stimulation of muscimol efflux by 50μM‐GABA, suggest that muscimol and GABA share some common transport carriers. Since L‐histidine inhibited muscimol uptake hut not GABA high affinity uptake, at least part of the observed muscimol uptake may be mediated by the small basic’amino acid transport system. Muscimol appeared to he taken up into nerve terminals, since uptake was inhibited by the neuronal uptake inhibitor cis‐3‐aminocyclohexanecarboxylic acid but not by the glial uptake inhibitor β‐alanine. Muscimol efflux was stimulated in a calcium‐dependent manner by an increased potassium ion concentration.

Effects of the Areca nut constituents arecaidine and guvacine on the action of GABA in the cat central nervous system.

Arecaidine and guvacine, constituents of the nut of Areca catechu, inhibited the uptake of GABA and beta-alanine, but not that of glycine, by slices of cat spinal cord. In cats anesthetised with pentobarbitone, electrophoretic arecaidine enhanced the inhibitory actions of GABA and beta-alanine, but not those of glycine or taurine, on the firing of spinal neurones. Similarly, electrophoretic guvacine enhanced the inhibition of spinal neurones by GABA but not that by glycine. The uptake of GABA by slices of cat cerebellum was inhibited by arecaidine, and the effect of electrophoretic GABA on the firing of cerebellar Purkinje cells was enhanced by electrophoretic arecaidine. When administered intravenously arecaidine failed to affect synaptic inhibitions considered to be mediated by GABA. Intravenous arecaidine had no effect on either spinal prolonged (presynaptic) inhibition (20mg/kg), dorsal root potentials (20mg/kg) or basket cell inhibition of Purkinje cells (250 mg/kg), although topical arecaidine (6.6-10 x 10(-3) M) blocked this latter inhibition. Large doses of arecaidine (1 g/kg subcutaneous) marginally reduced the lethal effects of bicuculline in mice but appeared to have little or no anticonvulsant activity.

Potentiation of L‐Glutamate and L‐Aspartate Excitation of Cat Spinal Neurones by the Stereoisomers of threo‐3‐Hydroxyaspartate

Active uptake may limit the action of L-glutamate and L-aspartate as excitatory transmitters in the mammalian C N S (Johnston, 1978). The excitation of ca t spinal neurones by electrophoretically administered L-glutamate and [--aspartate can be potentiated by similarly administered p-chloromercuriphenylsulphonate (Curtis e t al., 1970), a relatively non-specific inhibitor of amino acid uptake in slices of cat spinal cord (Balcar and Johnston, 1973) and rat brain (Curtis et al., 1970). The sectioning of pathways in the rat brain indicates an association between nerve terminals which probably release L-glutamate and a L-glutamateii-aspartate high-affinity uptake system (Divac et al., 1977); glial cells probably possess a similar uptake system (Roberts and Watkins, 1975). Threo-3hydroxy-L-aspartate (THLA) is an extremely potent and selective inhibitor of the high-affinity uptake of Lglutamate and L-aspartate in rat-brain slices, and its stereoisomer threo-3-hydroxy-~-aspartate (THDA) is only marginally less effective (Balcar e t al., 1977). In the present investigation these inhibitors have been found to increase the excitant actions of L-glutamate, L-aspartate, and D-glutamate on cat spinal neurones, with little effect on the excitant actions of acetylcholine, kainate, and N-meth yl-D-aspartate. Experiments were performed on three spinal cats anaesthetised with pentobarbitone sodium (35 mg/kg i.p.). Standard techniques were used for recording extracellular action potentials of lumbar dorsal horn interneurones and Renshaw cells using the centre barrel of seven-barrel glass micropipettes; test compounds were ejected electrophoretically from the outer barrels, which contained the following aqueous solutions: acetylcholine bromide (ACh, 250 mM), sodium L-aspartate, sodium L-glutamate, and sodium D-glutamate (each 200 mM, p H 7 . 9 , sodium kainate (25 mM in 125 mM-sodium chloride, p H 7.5), and the Land D-isomers of the lysine salts of threo-3hydroxyaspartate, prepared as described by Balcar et al. (1977) (THLA and THDA, 200 mM, p H 7.6). Both THLA and THDA excited neurones in the absence of excitation by other compounds, T H L A being some two to five times more active than THDA. THLA varied from being one-third to three times as potent as L-glutamate, and this wide variation in relative potency may indicate that THLA, and THDA to some extent, activate neurones through receptors other than those most sensitive to L-glutamate. The rate of recovery from the excitant effects of THLA was similar to that of Laspartate and L-glutamate. Under conditions where THLA and THDA had little or no effect on the spontaneous firing rate, excitation induced by the administration of L-glutamate was clearly potentiated. The difference in potency of the two isomers in enhancing the effect of L-glutamate was less than, and often the reverse of, that which might be expected if this effect were due simply to direct excitation and broadly consistent with that anticipated from uptake studies using rat-brain slices. T h e specificity of the potentiation by T H L A and THDA of amino acid excitation was examined using a variety of excitants chosen on the basis of their possible interaction with certain transport processes. L-Aspartate is as good a substrate as L-glutamate for the acidic amino acid high-affinity transport systems in rat-brain slices (Balcar and Johnston, 1 9 7 2 ~ ) and cat spinal cord (Balcar and Johnston, 1973), while D-glutamate is considered to be transported only by low-affinity systems (Benjamin and Quastel, 1976; Takagaki, 1976)1 Although a weak competitive inhibitor of L-glutamatek-aspartate highaffinity uptake in rat brain slices, kainate is not taken up by slices of rat brain or cat spinal cord (Johnston et al., 1979 and unpublished ohservutions). NMDA inhibits nei ther the highnor t h e low-affinity uptake of Lglutamate by rat brain slices and thus is not a substrate for either system (Balcar and Johnston, 19726). Acetylcholine does not influence the high-affinity uptake of L-glutamate by rat brain slices (Balcar and Johnston, 1972u), and extracellular hydrolysis in cat spinal tissue by acetylcholinesterase limits its apparent effectiveness when administered microelectrophoretically (Curtis and Eccles, 1958).

The dual effects of GABA and related amino acids on the electrical threshold of ventral horn group Ia afferent terminations in the cat

SummaryAmino acids were administered microelectrophoretically near the unmyelinated terminations of extensor muscle Ia afferent terminations stimulated electrically in the vicinity of lumbar motoneurones in anaesthetized cats. The predominant effect of one group (structurally related to GABA, poor substrates for in vitro amino acid uptake systems) was a reduction in the threshold (depolarization). The second group (including GABA and structural analogues which are substrates for GABA transport systems in vitro) had biphasic effects, an initial reduction being followed by an increase in threshold. The third group (structurally unrelated to GABA, substrates for amino acid uptake systems) only increased Ia termination thresholds.Reductions in termination thresholds, but not increases, were associated with diminution of synaptically evoked primary afferent depolarization, and were decreased by bicuculline methochloride. Many amino acids increased the electrical resistance of the intraspinal medium near the orifices of the barrels of seven barrel micropipettes, and for L-histidine, one of the third group of amino acids, both this effect and the increased threshold of terminations were reversibly modified by microelectrophoretic ouabain.These observations suggest that GABA-mimetics depolarize Ia terminations by interacting with bicuculline-sensitive receptors similar to those at hyperpolarizing gabergic synapses upon spinal interneurones. In addition, under the experimental conditions used, these and other amino acids increase termination thresholds, probably in the absence of any change in membrane conductance, an effect resulting from alterations in the ionic constitution of the extracellular medium around the orifices of micropipettes ejecting amino acids consequent upon the ouabain-sensitive co-transport of amino acids and sodium ions into neurones and glia.

The Synthesis and Activity of cis‐and trans‐2‐(Aminomethyl) cyclopropanecarboxylic Acid as Conformationally Restricted Analogues of GABA

Abstract: The synthesis of cis‐2‐(aminomethyl) cyclopropanecarboxylic acid, a new analogue of GABA in a folded conformation, is described, as is also an improved preparation of trans‐2‐(aminomethyl) cyclopropanecarboxylic acid. When adminstered microelectrophoretically the trans isomer was more potent than GABA as a bicuculline‐sensitive depressant of the firing of cat spinal neurons in vivo, whereas the cis‐isomer was less potent than GABA and its effects appeared not to be sensitive to bicuculline methochloride. Trans‐2‐(aminomethyl) cyclopropanecarboxylic acid was a weak inhibitor of the sodium‐dependent uptake of GABA by mini slices of rat cerebral cortex and a substrate for the GABA: 2‐oxoglutarate aminotransferase activity in extracts of rat brain mitochondria. The cis isomer did not influence GABA uptake or aminotransferase activity and neither isomer reduced glutamate decar‐boxylase activity in rat brain homogenates. Both cyclopropane isomers inhibited the sodium‐independent binding of GABA to synaptic membranes from rat brain and their relative potencies together with those found for the stereochemically related unsaturated derivatives, cis‐and trans‐4‐aminocrotonic acid, were broadly consistent with the activity observed for these compounds in vivo on cat spinal neurons. These studies reinforce the evidence that extended rather than folded conformations of GABA are active at most GABA recognition sites within the mammalian central nervous system.

Time course of GABA and glycine actions on cat spinal neurones: Effect of pentobarbitone

Abstract In unanaesthetized decerebrate cats the time course of recovery of the firing of spinal interneurones after electrophoretic glycine and GABA are virtually identical; systemically administered pentobarbitone increased that of GABA and had little or no effect on that of glycine. Such an action could be important in the enhancement and prolongation of GABA-mediated synaptic inhibition by pentobarbitone and other anaesthetics.