Tage Honore

Structure-activity relationships in the development of excitatory ammo acid receptor agonists and competitive antagonists

Development of new selective ligands for excitatory amino acid receptors has been fundamental in supporting this rapidly developing field. Some of the most important ligands have come from the laboratories of Jeff Watkins, Povl Krogsgaard-Larsen and Tage Honoré, who collaborate in this double-length review to describe the chemical features and SARs of agonists and antagonists, particularly those features associated with subtype selectivity.

Brain Glutamate Transporter Proteins Form Homomultimers

Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

Dopamine release in the nucleus caudatus and in the nucleus accumbens is under glutamatergic control through non-NMDA receptors: a study in freely-moving rats

Perfusion with quisqualate (5 x 10(-6) M) and kainate (5 x 10(-7) M), selective agonists of glutamate receptors, enhanced the release of dopamine in both caudate and accumbens nuclei of freely-moving rats, measured by the transcerebral microdialysis technique. In contrast, N-methyl-D-aspartate (NMDA) did not affect dopamine release, except at very high concentrations (10(-2) M). The quisqualate-kainate antagonist, FG 9041 (DNQX), antagonized the elevation of dopamine release induced by quisqualate and, furthermore, reduced that of kainate. CPP, a selective NMDA antagonist, did not counteract the quisqualate- or kainate-induced stimulation of dopamine release. The enhancement of dopamine release after quisqualate and kainate was accompanied by behavioural stimulation characterized by grooming, rearing, hypermotility with sniffing and confined sniffing. This behavioural syndrome could be blocked by haloperidol. Conversely, perfusion with NMDA did not activate behaviour even at high concentrations. These results indicate that the dopaminergic system, within the caudate and the accumbens nuclei, is under glutamatergic control through kainate and quisqualate receptors, while the NMDA receptors do not appear to be involved.

The Binding of [3H]AMPA, a Structural Analogue of Glutamic Acid, to Rat Brain Membranes

Abstract: Binding of [3H]AMPA to rat brain membranes was investigated. The binding was saturable and reversible at physiological pH. Computer‐aided Scatchard analysis of the binding data, as determined by using l‐glutamic acid (l‐GLU) to define nonspecific binding, suggested the presence of two independent binding sites, with KDs of 9 and 2440 nm, respectively. Additional freezing, thawing and washing sequences gave membranes with only one binding site, with a KD of 278 nm. [3H]AMPA binding exhibited the highest blevel in striatal membranes. A series of analogues of GLU and aspartic acid (ASP) were tested as inhibitors of [3H]AMPA binding. l‐ASP and compounds which interact predominantly with N‐methyl‐d‐aspartic acid (NMDA) receptor sites were inactive as inhibitors of [3H]AMPA binding, whereas l‐GLU and compounds which interact predominantly with glutamic acid diethyl ester receptor sites were inhibitors with the same order of potency as that shown by the excitatory action in vivo. The result suggests that [3H]AMPA might represent binding to an excitatory GLU receptor.

Evaluation of the β-carboline ZK 93 426 as a benzodiazepine receptor antagonist

We describe here biochemical and pharmacological effects of the β-carboline ZK 93426, a new and potent benzodiazepine (BZ) receptor antagonist. ZK 93426 was compared with Ro 15-1788 and CGS 8216, two compounds previously described as BZ receptor antagonists. Certain effects of ZK 93426, Ro 15-1788 and CGS 8216 were quite similar (e.g., 3H-FNM displacement, “GABA ratio”, “photo-shift”). In most pharmacological tests ZK 93426 and Ro 15-1788 lacked overt effects; Ro 15-1788 was a weak agonist in some paradigms, while ZK 93426 exhibited a potent proconflict effect but also a weak anticonvulsant effect. This interesting finding with ZK 93426 suggests that BZ receptor ligands may possess differential efficacy at BZ receptor subtypes. In contrast, CGS 8216 exhibited potent proconvulsant effects in several paradigms in addition to proconflict and pentylenetetrazol generalizing effects. ZK 93426, Ro 15-1788 and CGS 8216 were almost equally potent as antagonists of the effects of BZ receptor agonists, such as diazepam and lorazepam. However, ZK 93426 was the most potent inhibitor of the convulsions produced by the BZ receptor inverse agonist DMCM.

Synergism of the AMPA-antagonist NBQX and the NMDA-antagonist CPP with L-Dopa in models of Parkinson's disease

SummaryDegeneration of dopaminergic nigrostriatal neurons in Parkinsons disease results in an overactivity of excitatory glutamatergic projections from the subthalamic nucleus to the output nuclei of the basal ganglia resulting in rigidity and akinesia. In theory pharmacological blockade of these overactive systems should improve parkinsonian symptomatology. The selective AMPA-antagonist NBQX and the competitive NMDA-antagonist CPP are not effective in animal models of Parkinsons disease when given alone but ameliorate parkinsonian symptomatology and stimulate locomotor activity when co-administered with a threshold dose of L-Dopa. These synergistic effects are seen in the MPTP-treated (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) common marmoset and the rat with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra. Therefore competitive NMDA and non-NMDA antagonists may offer a new therapeutic strategy for the treatment of Parkinsons disease.

Chaotropic Ions Affect the Conformation of Quisqualate Receptors in Rat Cortical Membranes

Abstract: Binding of [3H](R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA) to quisqualate receptors in the presence of SCN ions produced curvilinear Scatchard plots. Kinetic investigations of [3H]‐AMPA binding showed that the curvilinearity cannot be explained by assuming binding to two separate binding sites or by considering it due to cooperative interaction. A more likely explanation is that the quisqualate receptors exist in two states, one with high and one with low affinity for [3H]AMPA. Chaotropic ions change the relaxation constant between the two states.

ZK 91296, a partial agonist at benzodiazepine receptors

ZK 91296 (ethyl 5-benzyloxy-4-methoxymethyl-β-carboline-3-carboxylate) is a potent and selective ligand for benzodiazepine (BZ) receptors. Biochemical investigations indicate that ZK 91296 may be a partial agonist at BZ receptors. Such partial agonism may explain to some extent why ZK 91296 needs higher BZ receptor occupancy than diazepam for the same effect against chemical convulsants and for behavioural effects. The lack of sedatiye effects, and the very potent inhibition of reflex epilepsy, spontaneous epilepsy and DMCM-induced seizures suggest, furthermore, that ZK 91296 may possess pharmacological selectivity for a particular type of BZ receptor interaction, perhaps including topographic as well as receptor subtype differentiation.

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non‐N‐methyl‐D‐aspartate (non‐NMDA) glutamate receptor antagonist [3H]6‐cyano‐7‐nitro‐quinoxaline‐2,3‐dione ([3H]‐CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD= 67 ± 9.0 nM and Bmax= 3.56 ± 0.34 pmol/mg protein. In displacement studies, quisqualate, L‐glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1= 9.0 ± 3.5 nM, Bmax= 0.15 ± 0.05 pmol/mg protein, KD2= 278 ± 50 nM, and Bmax= 1.54 ± 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L‐[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non‐NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.

Glutamate-Induced Increase in Intracellular Ca2+ in Cerebral Cortex Neurons Is Transient in Immature Cells but Permanent in Mature Cells

Abstract: The free cytosolic Ca2+ concentration ([Ca2+],) of cultured cerebral cortex neurons was determined using a fluorescent Ca2+chelator (Fluo‐3) after exposure of the neurons to glutamate. Mature neurons (8 days in culture) responded within 45 s to 100 μM glutamate by an increase in [Ca2+]j from 75 to 340 nM, an increase that during the following 6 min of exposure reached 400 nM. This increase in [Ca2+]j could not be reversed by removal of glutamate. In the absence of extracellular CaCl2, only part of the initial, rapid, glutamate‐induced increase in [Ca2+]j was observed in these neurons. In contrast to these findings, neurons cultured for only 2 days (immature neurons) exhibited only a small (from 75 to 173 nM) increase in [Ca2+]j after exposure to 100 μM glutamate, and this rapid increase in [Ca2+]j tended to decline on prolonged exposure to glutamate. Moreover, after removal of glutamate, the increase in [Ca2+]j was fully reversible. Pharmacological characterization of the response to glutamate in mature neurons showed that the yN‐methyl‐D‐aspartate (NMDA) receptor antagonists phencyclidine and D‐2‐amino‐5‐phosphonovalerate blocked 75 and 90%, respectively, of the response, whereas the non‐NMDA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione had little effect.