Nicholas J. Toms

The effects of (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine ((RS)‐CPPG), a potent and selective metabotropic glutamate receptor antagonist

1 In this study we describe the potent antagonist activity of a novel metabotropic glutamate (mGlu) receptor antagonist (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine ((RS)‐CPPG) which exhibits selectivity for mGlu receptors (group II and III) negatively coupled to adenylyl cyclase in the adult rat cortex. 2 Both the L‐2‐amino‐4‐phosphonobutyrate (L‐AP4) and (2S, 1′S, 2′S)‐2‐(carboxycyclopropyl)glycine (L‐CCG‐1) inhibition of forskolin‐stimulated cyclic AMP accumulation were potently reversed by (RS)‐CPPG (IC50 values: 2.2 ± 0.6 nM and 46.2 ± 18.2 nM, respectively). 3 In contrast, (RS)‐CPPG acted as a weak antagonist against group I mGlu receptors. In neonatal rat cortical slices, (RS)‐CPPG antagonized (KB = 0.65 ± 0.07 mM) (1S, 3R)‐1‐aminocyclopentane‐1, 3‐dicarboxylic acid ((1S, 3R)‐ACPD)‐stimulated phosphoinositide hydrolysis. (RS)‐CPPG (100 μm) failed to influence L‐quisqualate‐stimulated phosphoinositide hydrolysis in cultured cerebellar granule cells. 4 In the rat cerebral cortex, (RS)‐CPPG is the most potent antagonist of group II/III mGlu receptors yet described (with 20 fold selectivity for group III mGlu receptors), having negligible activity at group I mGlu receptors.

A novel kainate receptor ligand [3H]-(2S,4R)-4-methylglutamate : Pharmacological characterization in rabbit brain membranes

Since kainate evokes large non-desensitizing currents at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate is of limited use in discriminating between AMPA and kainate receptors. Following recent reports that (2S,4R)-4-methylglutamate is a kainate receptor-selective agonist, we have radiolabelled and subsequently characterized the binding of [3H]-(2S,4R)-4-methylglutamate to rabbit whole-brain membranes. [3H]-(2S,4R)-4-methylglutamate binding was rapid, reversible and labelled two sites (KD1 = 3.67+/-0.50 nM/Bmax1 = 0.54+/-0.03 pmol/mg protein and KD2 = 281.66+/-12.33 nM/ Bmax2 = 1.77+/-0.09 pmol/mg protein). [3H]-(2S,4R)-4-methylglutamate binding was displaced by several non-NMDA receptor ligands: domoate > kainate >> L-quisqualate > or = L-glutamate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) >> (S)-AMPA = (S)-5-fluorowillardiine > NMDA. Neither the metabotropic glutamate receptor agonists (1S,3R)-ACPD or L-AP4, together with the L-glutamate uptake inhibitor L-trans-2,4-PDC, influenced binding when tested at 100 microM. We conclude that [3H]-(2S,4R)-4-methylglutamate is a useful radioligand for labelling kainate receptors. It possesses high selectivity, and possesses a pharmacology similar to that for rat cloned low-affinity (Glu5 and 6) kainate receptor subunits.

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.