D. E. Jane

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.

Potent antagonists at the L-AP4- and (1S,3S)-ACPD-sensitive presynaptic metabotropic glutamate receptors in the neonatal rat spinal cord

In this report we describe the actions of two novel compounds, (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) and (S)-alpha-ethylglutamate (EGLU), which are potent antagonists at two types of presynaptic metabotropic glutamate (mGlu) receptors in the neonatal rat spinal cord. Selective activation of these receptors by L-2-amino-4-phosphonobutyrate (L-AP4) or (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3S)-ACPD) results in the depression of the monosynaptic component of the dorsal root-evoked ventral root potential (DR-VRP). CPPG produces rightward parallel shifts of the dose-response curves for both L-AP4- and (1S,3S)-ACPD, with Schild slope in each case close to unity, consistent with a competitive mechanism of antagonism. CPPG is the most potent antagonist yet described for both L-AP4- and (1S,3S)-ACPD-sensitive presynaptic mGlu receptors but displays a 30-fold selectivity for the L-AP4-sensitive receptor over the (1S,3S)-ACPD-sensitive receptor (KD values 1.7 microM and 53 microM, respectively). EGLU, on the other hand, is selective for the (1S,3S)-ACPD-sensitive receptor, displaying little or no activity at the L-AP4-sensitive site. EGLU produces a rightward parallel shift of the dose-response curve to (1S,3S)-ACPD, with Schild slope close to unity, again indicative of a competitive mode of antagonism (KD 66 microM). Both CPPG and EGLU displayed only weak or no antagonist activity at postsynaptic metabotropic and ionotropic glutamate receptors.

New phenylglycine derivatives with potent and selective antagonist activity at presynaptic glutamate receptors in neonatal rat spinal cord

The depression of the monosynaptic excitation of neonatal rat motoneurones produced by the metabotropic glutamate receptor (mGluR) agonists (1S,3S)-1-aminocyclopentane-1, 3-dicarboxylate (ACPD) or L-2-amino-4-phosphonobutyrate (L-AP4) was antagonized by three novel phenylglycine analogues: (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG), (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) and (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG). The potencies of all the new compounds were greater than that of the previously reported (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG). For L-AP4-sensitive presynaptic mGluRs, the order of antagonist potency found was MPPG > MSPG > MTPG > MCPG. In contrast, the order of antagonist potency found for (1S,3S)-ACPD-sensitive presynaptic mGluRs was MTPG > MPPG > MSPG > MCPG. To date, MPPG (KD 9.2 microM) is the most potent L-AP4-sensitive receptor antagonist yet tested on the neonatal rat spinal cord. In addition, MTPG (KD 77 microM) is the most potent antagonist yet tested for (1S,3S)-ACPD-sensitive receptors in this preparation.

Structure-activity relationships of new agonists and antagonists of different metabotropic glutamate receptor subtypes

1 We investigated the agonist and antagonist activities of 22 new phenylglycine and phenylalanine derivatives for metabotropic glutamate receptors (mGluRs) by examining their effects on the signal transduction of mGluR1, mGluR2 and mGluR6 subtypes expressed in Chinese hamster ovary cells. This analysis revealed several structural characteristics that govern receptor subtype specificity of the agonist and antagonist activities of phenylglycine derivatives. 2 Hydroxyphenylglycine derivatives possessed either an agonist activity on mGluR1/mGluR6 or an antagonist activity on mGluR1. 3 Carboxyphenylglycine derivatives showed an agonist activity on mGluR2 but an antagonist activity on mGluR1. 4 α‐Methylation or α‐ethylation of the carboxyphenylglycine derivatives converts the agonist property for mGluR2 to an antagonist property, thus producing antagonists at both mGluR1 and mGluR2. 5 Structurally‐corresponding phenylalanine derivatives showed little or no agonist or antagonist activity on any subtypes of the receptors. 6 This investigation demonstrates that the nature and positions of side chains and ring substituents incorporated into the phenylglycine structure are critical in determining the agonist and antagonist activities of members of this group of compounds on different subtypes of the mGluR family. 7 We also tested two α‐methyl derivatives of mGluR agonists. (2S, 1′S, 2′S)‐2‐(2‐Carboxycyclopropyl)glycine (L‐CCG‐I) is a potent agonist for mGluR2 but α‐methylation of this compound changes its activity to that of an mGluR2‐selective antagonist. In contrast, α‐methylation of L‐2‐amino‐4‐phosphonobutyrate (L‐AP4) results in retention of an agonist activity on mGluR6. Thus, α‐methylation produces different effects, depending on the chemical structures of lead compounds and/or on the subtype of mGluR tested.

α-methyl derivatives of serine-O-phosphate as novel, selective competitive metabotropic glutamate receptor antagonists

The antagonist selectivity and potency of two novel serine-O-phosphate derivatives (RS)-alpha-methylserine-O-phosphate (MSOP) and the monophenylester (RS)-alpha-methylserine-O-phosphate monophenyl-phosphoryl ester (MSOPPE) was investigated against L-2-amino-4-phosphonobutyrate (L-AP4)- and (1S,3S)-1-aminocyclopentane-1, 3-dicarboxylate (ACPD)-induced depressions of the monosynaptic excitation of neonatal rat motoneurones, mediated via metabotropic glutamate receptors (mGLuRs). MSOP was shown to be a selective antagonist for the L-AP4-sensitive presynaptic mGluR, displaying an apparent KD of 51 microM, compared to > 700 microM for the (1S,3S)-ACPD-sensitive presynaptic mGluR. In contrast, MSOPPE displayed antagonist activity at both presynaptic mGluR, with a three times greater selectivity for the (1S,3S)-ACPD-sensitive receptor over the L-AP4-sensitive mGluR (apparent KD values 73 microM and 221 microM, respectively). Therefore, on addition of an alpha-methyl group to the mGluR agonist serine-O-phosphate, we have developed an mGluR antagonist which is selective for the presynaptic L-AP4-sensitive receptor. In contrast, monoesterification of MSOP to give the monophenylphosphoryl ester (MSOPPE), confers a degree of selectivity for the (1S,3S)-ACPD-over the L-AP4-sensitive presynaptic mGluR. Neither MSOP nor MSOPPE had any activity on either postsynaptic mGLuRs or ionotropic receptors.

Structure-activity relationships for a series of phenylglycine derivatives acting at metabotropic glutamate receptors (mGluRs)

1 The actions of a series of twelve phenylglycine derivatives at metabotropic glutamate receptors (mGluRs) linked to both phosphoinositide hydrolysis (PI) and cyclic AMP were investigated. 2 PI hydrolysis was determined by the accumulation of [3H]‐inositol‐monophosphate ([3H]‐IP1) in neonatal rat cortical slices prelabelled with [3H]‐myo‐inositol. The non‐selective mGluR agonist (1S,3R)−1 ‐ aminocyclopentane − 1,3 ‐ dicarboxylic acid ((1S,3R)‐ACPD) produced a concentration‐dependent increase in [3H]‐IP1 (EC50 XXXX 20 μm). This agonist was subsequently used to investigate potential antagonist activity of the phenylglycine derivatives. Of the compounds tested (+)‐α‐methyl‐4‐ carboxyphenylglycine (M4CPG) and (RS)‐α‐ethyl‐4‐carboxyphenylglycine (E4CPG) were the most active with Kb values of 0.184 XXXX 0.04 mM and 0.367 XXXX 0.2 mM respectively. 3 Activity at adenylyl cylase‐coupled mGluRs was investigated by determining the accumulation of [3H]‐cyclic AMP in adult rat cortical slices. [3H]‐cyclic AMP accumulation, elicited by 30 μm forskolin, was inhibited by (2S,3S,4S)‐α‐(carboxycyclopropyl)glycine (L‐CCG‐1) and L‐2‐amino‐4‐phosphonobutanoate (L‐AP4) with respective EC50 values of 0.3 μm and 10 μm. Neither agonist was able to inhibit completely forskolin stimulated cyclic AMP accumulation; this is evidence that not all adenylyl cyclase is susceptible to modulation by mGluRs. Phenylglycine derivatives were examined for their ability to antagonize the inhibition of [3H]‐cyclic AMP accumulation by L‐CCG‐1 or L‐AP4 at their EC50 concentrations. 4 A rank order of potency of the phenylglycine derivatives as antagonists of L‐AP4 and L‐CCG‐1 was obtained. The most effective compound, (RS)‐α‐methyl‐3‐carboxymethylphenylglycine (M3CMPG) had IC50 values in the order of 1 μm against L‐AP4 and 0.4 μm against L‐CCG‐1. 5 The results from this study indicate that phenylglycine‐derived compounds can discriminate between groups of metabotropic glutamate receptors and may also display some selective activity between subtypes within groups. Future work based on these findings may lead to the development of more selective and potent compounds as important pharmacological tools.

Characterization of metabotropic glutamate receptor‐stimulated phosphoinositide hydrolysis in rat cultured cerebellar granule cells

1 The pharmacology of excitatory amino acid (EAA)‐stimulated phosphoinositide (PI) hydrolysis, monitored via [3H]‐inositol monophosphate accumulation, was investigated in primary cultures of rat cerebellar granule cells. 2 EAA‐stimulated PI hydrolysis peaked after 4–5 days in vitro and subsequently declined. 3 The agonist order of potency was found to be (EC50): L‐quisqualic acid (Quis) (2 μm)»L‐glutamate (50 μm)>(1S,3R)‐11‐aminocyclopentane‐1,3‐dicarboxylic acid ((1S,3R)‐ACPD) (102 μm). L‐Glutamate (Emax = 873% of basal activity) elicited the largest stimulation of PI hydrolysis, whereas Quis (Emax = 603%) and (1S,3R)‐ACPD (Emax = 306%) produced somewhat lower stimulations. 4 Several phenylglycine derivatives were found to be active in inhibiting 2 μm Quis‐stimulated PI hydrolysis, in order of potency (IC50): (S)‐4‐carboxy‐3‐hydroxyphenylglycine (41 μm) ≤ (S)‐4‐carboxyphenylglycine (51 μm)»(+)‐α‐methyl‐4‐carboxyphenylglycine (243 μm). 5 Cultured cerebellar granule cells of the rat appear to have Group I mGluR pharmacology similar to that reported for cloned mGluRl and provide an ideal system for investigating novel mGluRl ligands in a native environment.

Two Phenylglycine Derivatives Antagonize Responses to l-AP4 in ON Bipolar Cells of the Amphibian Retina

Light responses of retinal ON bipolar cells are mediated by metabotropic glutamate receptors selectively activated by L-2-amino-4-phosphonobutyric acid (L-AP4). Antagonists to L-AP4 receptors in ON bipolar cells have not previously been identified. This study examines the electrophysiological effects of (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), (RS)-4-4-chloro-3,5-dihydroxyphenylglycine (CDHPG) and (RS)-3,4,5-trihydroxyphenylglycine (THPG), at L-AP4 receptors in ON bipolar cells of the amphibian retina. Unlike its actions in spinal cord, in retinal ON bipolar cells MAP4 is a weak agonist which exhibits no detectable antagonism to L-AP4. On the other hand, CDHPG exhibits a mixture of agonist and antagonist properties. Addition of Co2+ and oxygenation of CDHPG turns the solution brown and enhances antagonist effects, suggesting that the antagonism reflects actions of a breakdown product of CDHPG. Although THPG did not prove to be this breakdown product, it also has electrophysiological effects consistent with an L-AP4 receptor antagonist. The results suggest that THPG and breakdown products of CDHPG may be antagonists to L-AP4 receptors in retinal ON bipolar cells, although the possibility that these compounds antagonize effects of L-AP4 by acting at some site in the transduction pathway of L-AP4 receptors cannot yet be excluded.