Discovery and characterization of non-competitive antagonists of group I metabotropic glutamate receptors.

We have investigated the mechanism of inhibition of the new group I mGluR antagonists CPCCOEt and MPEP and determined that both compounds have a non-competitive mode of inhibition. Furthermore using chimeric/mutated receptors constructs we have found that these antagonists act at a novel pharmacological site located in the trans-membrane (TM). Specific non-conserved amino acid residues in the TM domain have been identified which are necessary for the inhibition by CPCCOEt and MPEP of the mGlul and mGlu5 receptors, respectively. Using molecular modeling a model of the TM domain was built for both mGlu1 and mGlu5 receptor subtypes. Docking of CPCCOEt and MPEP into their respective model allowed the modelisation of the novel binding site.

The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors.

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [(3)H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [(3)H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.

Similar potency of carbamazepine, oxcarbazepine, and lamotrigine in inhibiting the release of glutamate and other neurotransmitters.

We compared the effects of the antiepileptic drugs carbamazepine, oxcarbazepine, and lamotrigine on the release from rat brain slices of endogenous glutamate, [3H]-GABA, and [3H]-dopamine, elicited by the Na+ channel opener, veratrine, and of the same transmitters as well as [3H]-noradrenaline, [3H]-5-hydroxytryptamine, and [3H]-acetylcholine, elicited by electrical stimulation. The three antiepileptic drugs inhibited veratrine-induced release of endogenous glutamate, [3H]-GABA, and [3H]-dopamine, with IC50 values between 23 and 150 microM, in or near the concentration range in which they interact with Na+ channels, and there was little difference between the compounds. They were five to seven times less potent in inhibiting electrically as compared with veratrine-stimulated release of [3H]-GABA and [3H]-dopamine; similarly, carbamazepine and tetrodotoxin were more potent in inhibiting veratrine-induced as compared with electrically induced release of endogenous glutamate. Carbamazepine, oxcarbazepine, and lamotrigine also inhibited electrically stimulated release of [3H]-5-hydroxytryptamine (IC50 values, 150 to 250 microM) and [3H]-acetylcholine (IC50 values, 50 to 150 microM); [3H]-noradrenaline release was affected to a lesser degree. The active concentration ranges of these drugs with respect to inhibition of veratrine-stimulated neurotransmitter release matched the therapeutic plasma and brain concentrations. It is uncertain whether these effects are relevant in vivo at anticonvulsant doses, because the drugs are markedly less potent in inhibiting the more physiologic release elicited by electrical stimulation. Therefore, the hypothesis that inhibition of glutamate release is the mechanism of anticonvulsant action of lamotrigine (or carbamazepine and oxcarbazepine) is doubtful. Other consequences of Na+ channel blockade may have an important role.

Investigations on GABAB receptor-mediated autoinhibition of GABA release.

In this study, we have investigated the effects of phaclofen on the [3H] overflow from [3H]GABA prelabelled rat cortical slices and its interaction with the effects of (-)-baclofen in dependence of the stimulation frequency. (-)-Baclofen strongly depressed the [3H] overflow in the frequency range of 0.125 to 4 Hz to a constant residual level (IC50 = 0.37 micromol/l at 0.125 Hz), but became inactive above. The potency of the (+)-enantiomer was considerably weaker by a factor of nearly 1000. The GABAB antagonist, phaclofen, increased [3H] overflow at 300 micromol/l and, more-markedly, at 3 and 1 mmol/l, respectively. However, the increase was virtually independent of the frequency between 0.125 and 16 Hz. If the compound interacted only with the putative GABAB autoreceptor involved in the regulation of GABA release, the extent of the enhancing effect should increase with increasing frequency because of the concomitant rise in synaptic GABA concentration. In order to further investigate this phenomenon, the IC50 of (-)-baclofen and antagonism of phaclofen against (-)-baclofen were determined at 0.125 Hz and 2 Hz, respectively. Whereas the IC50 of (-)-baclofen was 0.63 +/- 0.04 micromol/l at 0.125 Hz, it increased to 4.88 +/- 0.45 micromol/l at 2 Hz. The pA10-values of phaclofen were about the same at both frequencies, whereas the pA2-values differed by a factor of 2.3.(ABSTRACT TRUNCATED AT 250 WORDS)