R.H. Evans

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.

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.

Antagonism of excitatory amino acid-induced responses and of synaptic excitation in the isolated spinal cord of the frog.

1 A range of compounds has been tested for excitatory amino acid agonist or antagonist activity and for effects on synaptic activity on isolated hemisected spinal cords of frogs. 2 l‐Monoamino dicarboxylic acids of chain length up to 8 carbon atoms (l‐α‐aminosuberate) were all agonists. 3 Within a series of d‐monoamino dicarboxylic acids, and with diamino dicarboxylic acids (mainly unresolved mixtures of diasteroisomers), there was a progression from agonist activity, for compounds of chain length equal to or shorter than glutamate, to antagonist activity, for compounds of longer chain length. d‐α‐Aminosuberate (DαAS) was the most potent antagonist. 4 The antagonist actions of these substances showed a Mg2+ ‐like selectivity with respect to depolarizations produced by different excitants. N‐methyl‐d‐aspartate (NMDA) was the most susceptible agonist and quisqualate and kainate the least susceptible. Responses to other excitatory amino acids, including l‐glutamate and l‐aspartate, showed intermediate sensitivity to the antagonists. 5 A parallelism was observed between the relative potencies of mono‐ and diamino dicarboxylic acids as NMDA antagonists and their relative potencies as depressants of synaptic responses. 6 The results support the concept of different types of excitatory amino acid receptors, with NMDA and its antagonists acting predominantly on one type. These NMDA receptors are probably transmitter receptors activated by an excitatory amino acid transmitter.

Selective antagonism by Mg2+ of amino acid-induced depolarization of spinal neurones

In the isolated frog or rat spinal cord, low concentrations of Mg2+ (0.5–1.00 mM) markedly depress, in a substantially Ca2+-independent manner, ventral root depolarizations produced by dorsal root stimulation and by certain amino acids (e. g. N-methyl-D-aspartate and L-homocysteate) but do not depress depolarizations produced by other excitatory amino acids (e. g. kainate and quisqualate). L-Aspartate-induced depolarizations are more sensitive to Mg2+ then are L-glutamate-induced depolarizations.

Potentiation of the effects of GABA by pentobarbitone.

Pentobarbitone (50 microM) has been shown to potentiate GABA-induced responses in rat isolated superior cervical ganglia and in dorsal or ventral root fibres of immature rat isolated spinal cords. Measurements of dose ratios for the antagonism of GABA-induced responses of dorsal root fibres by bicuculline showed that pentobarbitone (50 microM) did not significantly alter this antagonism. This result indicates that the antagonism of GABA by bucuculline at dorsal root fibres is not reversed by pentobarbitone. Ventral root responses to GABA (but not glycine) were also potentiated by pentobarbitone (+/-)-Nipecotic acid (300 microM) potentiated responses to GABA much more than those to 3-aminopropane-sulphonic acid.

Effect of 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) on dorsal root-, NMDA-, kainate- and quisqualate-mediated depolarization of rat motoneurones in vitro

1 Mature in vitro rat spinal cord preparations have been used to compare the depressant effects of 6‐cyano‐2,3‐dihydroxy‐7‐nitroquinoxalinedione (CNQX) and kynurenate on transmission from low threshold myelinated primary afferents in dorsal roots. EC50 values ± s.e.mean (number of preparations in parentheses) for depression of the monosynaptic ventral root reflex were respectively 1.0 ± 0.3 μm (5) and 135 ± 15μm (3) for CNQX and kynurenate. Transmission through superior cervical ganglia was not significantly affected by concentrations of CNQX up to 100 μm or kynurenate up to 5 mm. 2 Immature in vitro rat spinal cord preparations were used to measure dose‐ratios for antagonism of depolarizations induced by N‐methyl‐d‐aspartate (NMDA), kainate or quisqualate by 4, 10 and 25 μm CNQX. In the presence of 0.75 mm Mg2+ pA2 values + s.e.mean were respectively 4.62 ± 0.05 (16), 5.79 ± 0.01 (4) and 5.59 ± 0.05 (16) for each agonist. These values were not significantly altered in the absence of added Mg2+. The mean pA2 values for kainate were significantly higher than those for quisqualate (P < 0.01). 3 Antagonism of NMDA‐induced depolarizations was evident at 10 and 25 but not 4 μm CNQX. The antagonism of NMDA was reversed by d‐serine (100 and 200 μm). 4 A similarity between the relative potencies of both CNQX and kynurenate for depression of synaptic transmission and antagonism of amino acid‐induced depolarizations indicates that monosynaptic transmission from myelinated primary afferents to motoneurones is mediated by kainate and/or quisqualate sub‐types of non‐NMDA receptors.

Specific antagonism of excitant amino acids in the isolated spinal cord of the neonatal rat.

The specificity of the neurodepressant actions of D-alpha-aminoadipate, alpha,epsilon-diaminopimelic acid, HA-966 (HAP) and Mg2+ has been investigated. On the isolated spinal cord of the neonatal rat, ventral root depolarizations produced by kainate, substance P, carbachol and noradrenaline were relatively unaffected by the same concentrations (0.25--1 mM) of the agents as those which reduced synaptic activity and ventral root depolarizations produced by N-methyl-D-aspartate (especially), L-aspartate and L-glutamate. The same or higher concentrations of the agents did not affect excitatory transmission in the isolated rat superior cervical ganglion. It is proposed that the agents specifically block synaptic transmission mediated by an excitatory amino acid.

GABA-mimetic action of etomidate.

A comparison of antagonism by bicuculline or strychnine of the effects of GABA or etomidate on rat isolated superior cervical ganglia, frog isolated hemisected spinal cords and rat central neurones in vivo indicates that etomidate has GABA-mimetic actions.

The pharmacological selectivity of three NMDA antagonists

Three N-methyl-D-aspartate (NMDA) antagonists (+/-)2-amino-5-phosphonopentanoate (AP5), 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and ((+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a.d.)cyclohepten-5,10- imin e maleate) (MK-801) have been tested for selectivity against depolarization of motoneurones induced by carbachol, 5-hydroxytryptamine, noradrenaline and substance P in isolated immature rat spinal cord preparations. AP5 (400 microM) and CPP (50 microM) gave mean dose-ratios, for antagonism against NMDA, of 103 +/- 14.9 S.E.M. (eight preparations) and 34.1 +/- 1.9 S.E.M. (14 preparations). MK-801 (1 and 10 microM) was the most potent of the three antagonists yielding dose ratios greater than 100 after 120 min treatment. MK-801 potentiated responses induced by 5-hydroxytryptamine and noradrenaline given dose-ratios of 0.22 +/- 0.16 S.E.M. and 0.20 +/- 0.06 S.E.M., respectively (four preparations). The three antagonists produced no significant antagonism of the non-amino acid agonists (four preparations for each agonist) when dose-ratios against NMDA were at least 40. The observations support the use of these antagonists as tools to identify sites of excitatory amino acid-mediated transmission.

The depressant action of baclofen on the isolated spinal cord of the neonatal rat

Neurotransmission in isolated hemisected spinal cord preparations from immature rats was depressed by micromolar levels of baclofen (threshold 0.5 microM). The depressant action of baclofen was not antagonised by bicuculline and baclofen, unlike GABA, did not depolarize primary afferent fibres. Neurotransmission in isolated vas deferens, anococcygeus muscle and superior cervical ganglion of the rat was unaffected by baclofen (0.1-1 mM). Depolarization of motoneurones, as recorded in ventral roots of tetrodotoxin-blocked spinal cord preparations, induced by excitant amino acids, substance P, noradrenaline or carbachol was unaffected by baclofen (250 microM or higher). The depressant action of baclofen on spinal cord preparations was similar to that produced by the excitant amino acid antagonist alpha,epsilon-diaminopimelic acid. A structure-activity study showed that the (--)-isomer of baclofen was over 20 times more potent than the (+)-isomer as a spinal depressant. Also the position and nature of the halogen substitutent in the ring is critical with baclofen giving optimal activity. It is concluded that the depressant action of baclofen from depression of the presynaptic release of excitant amino acid transmitter(s).