Cyclothiazide binding to the GABAA receptor.

In order to explore the molecular interaction between cyclothiazide (CTZ) and gamma-aminobutyric acidA (GABAA) receptors, possibly underlying inhibition of GABAA receptor currents, [3H]-CTZ was synthesized. Binding of [3H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (-)[1S,9R]-bicuculline methiodide (BMI) (EC(50,BMI)=500+/-80microM). GABA decreased [(3)H]-CTZ binding induced by the presence 300microM and 3mM BMI with IC(50,GABA) values of 300+/-50microM and 5.0+/-0.7mM, respectively. Binding of CTZ to [3H]-CTZ labeled sites was characterized by IC(50,CTZ) values of 0.16+/-0.03muM ([BMI]=300microM) and 7.0+/-0.5microM ([BMI]=3mM). Binding of the diastereomeric fraction [3H]-(3R,1'S,4'S,5'R+3S,1'R,4'R,5'S)-CTZ induced by 3mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC(50,CTZ)=80+/-15nM and IC(50,GABA)=13+/-3mcapital EM, Cyrillic. In the absence of BMI, CTZ (1mM) significantly decreased GABA-induced enhancement of [3H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding.

Suppression of neuronal network excitability and seizure-like events by 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine in juvenile rat hippocampus: involvement of a metabotropic glutamate receptor.

We present data on the antiepileptic potency of 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) in juvenile (P9-13) rat hippocampal slices and in particular Q5's action mechanism and target. Q5 (200-500 microM), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/Kainate receptor antagonists blocked low-[Mg2+]-induced seizure-like events (SLE) in the CA3 region. Q5 (100 microM) decreased Glu-induced [35S]guanosine 5'-O-(3-thiotriphosphate) binding enhancement in brain homogenates, without interaction with ionotropic Glu receptor sites and Glu transport. In voltage-clamped CA3 pyramidal cells, Q5 (500 microM) depressed activities of spontaneous excitatory and inhibitory postsynaptic currents without affecting miniature inhibitory currents. Metabotropic Glu receptor (mGluR) subtype antagonists affected network excitability dissimilarly. Intracellular Ca2+ ion transients induced by the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were suppressed by Q5. Agreeing predictions obtained by modelling Q5 binding to different experimental conformations of mGlu1, Q5 was bound partially to an mGluR binding site in the presence of 1mM ACPD. Findings suggest the apparent involvement of a novel phenotype of action or a new mGluR subtype in the specific suppression of epileptiform activity by Q5 through the depression of network excitability.

Ca2+ ion accumulation precedes formation of O2-* in isolated brain mitochondria.

The relationship between mitochondrial Ca2+ accumulation and reactive oxygen species formation is studied by means of fast kinetics with fluorescence detection. Succinate or elevated [Na+] and [Ca2+] provided alternative conditions for induction of reactive oxygen species in isolated rat cerebrocortical mitochondria. Depending on Na+/Ca2+ exchanger and superoxide dismutase activities, formation of reactive oxygen species in mitochondria exposed to elevated [Na+] and [Ca2+] levels was characterized by t(1/2) = 12.3+/-2.9 ms. Production of reactive oxygen species by xanthine oxidase followed a similar time course. Both the processes were blocked by para-benzoquinone, indicating the primary formation of O2-*. Substantial increase in mitochondrial O2-*. was observed at [Ca2+] > or = 2.5 microM. Mitochondrial Ca2+ accumulation obeyed similar [Ca2+] dependence but shorter t(1/2) (2.5+/-0.3 ms), providing kinetic evidence for Ca2+ accumulation-dependent primary formation of O2-* in brain mitochondria.

Interaction of novel condensed triazine derivatives with central and peripheral type benzodiazepine receptors: synthesis, in vitro pharmacology and modelling.

Structurally related sets of triazino-quinoline, triazino-isoquinoline and pyrido-triazine derivatives were synthesised and their binding interactions with central (CBR)- and peripheral-type (PBR) benzodiazepine binding sites have been characterised. Of 33 compounds tested, a new compound, 2-(4-methylphenyl)-3H- [1,2,4] triazino [2, 3-a] quinolin-3-one (1 g) showed the lowest CBR binding inhibition constant (K(i) = 42 +/- 9 nM) and the highest CBR over PBR selectivity (>1300). All but the 4-methylphenyl (1 g) structural modifications decreased the affinity and selectivity of the parent compound, 2-phenyl-3H- [1,2,4]triazino[2,3-a]quinolin-3-one (1d) (K(i) = 69 +/- 9 nM, selectivity >890). Molecular interactions between selected ligands (standards and triazine derivatives) and alpha(1)gamma(2) subunit-interface residues in a GABA(A) receptor extracellular domain homology model have been calculated. Comparing data with calculations confirmed hydrogen bonding to gamma(2)Thr142 and hydrophobic interaction with alpha(1)His101 as being essential for high-affinity CBR binding.

Novel secoergoline derivatives inhibit both GABA and glutamate uptake in rat brain homogenates: synthesis, in vitro pharmacology, and modeling.

Three of twelve secoergoline derivatives (Z ethyl 4-[(ethoxycarbonylmethyl)methylamino]-2-methyl-3-phenylpent-2-enoate, 8; ethyl 1,6-dimethyl-3-oxo-5-phenyl-1,2,3,6-tetrahydropyridine-2-carboxylate, 9; Z methyl 4-[(methoxycarbonylmethyl)methylamino)-2-methyl-3-phenylpent-2-enoate, 11), containing bioisosteric sequences of GABA and Glu, inhibited both GABA and Glu transport through cerebrocortical membranes specifically. Compounds 8, 9, and 11 appeared to be equipotent inhibitors of GABA and Glu transport with IC50 values between 270 and 1100 microM, whereas derivatives 1-7, 10, and 12 were without effects. In the presence of GABA and Glu transport-specific nontransportable inhibitors, inhibition of GABA and Glu transport by 8, 9, and 11 proceeded in two phases. The two phases of inhibition were characterized by IC50 values between 4 and 180 nM and 360-1020 microM and different selectivity sequences. These findings may indicate the existence of some mechanism possibly mediated by a previously unrecognized GABA-Glu transporter. Derivatives with the cis, but not the trans configuration of bulky ester groups (8 vs 7 and 11 vs 12) showed significant inhibitory effect (IC50 values of 270 microM vs >>1000 microM and 1100 microM vs >>1000 microM on GABA transport, respectively). The cis-trans selectivity can be explained by docking these secoergolines in a three-dimensional model of the second and third transmembrane helices of GABA transporter type 1.

Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates.

Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites.

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.

GABA(B) receptor antagonist CGP-36742 enhances somatostatin release in the rat hippocampus in vivo and in vitro.

Here, we show the modulation of somatostatin functions in the hippocampus by the orally active 'cognition enhancer' GABA(B) receptor antagonist, (3-aminopropyl)n-butylphosphinic acid (CGP-36742), both in vivo and in vitro. Using high-pressure liquid chromatography-coupled electrospray mass spectrometry, we measured a two-fold increase in the extracellular level of somatostatin to CGP-36742 application in the hippocampus of anaesthetised rats. The basal release of [125I]somatostatin in the synaptosomal fraction was increased by CGP-36742 in concentrations lower than 1 muM. Simultaneous measurement of [14C]Glu and [3H]gamma-aminobutyric-acid ([3H]GABA) showed that CGP-36742 increased their basal release. However, prior [125I]somatostatin application suppressed the increase in the basal release of [14C]Glu and induced a net decrease in the basal release of [3H]GABA. Somatostatin application had a similar effect. In slices, CGP-36742 increased the postsynaptic effect of somatostatin on CA1 pyramidal cells. These results suggest a pre- and postsynaptic functional 'cross-talk' between coexisting GABA(B) and somatostatin receptors in the rat hippocampus.

[EFfect of quinazolone-alkyl-carboxylic acid derivatives on the transmembrane Ca2+ ion flux mediated by AMPA receptors]. / Kinazolon-alkil-karbonsav származékok hatása az alpha-amino-3-hidroxi-5-metil-4-izoxazol propionsav (AMPA) receptor által szabályozott transzmembrán Ca2+ ion fluxusra.

The excitatory neurotransmitter, Glu, plays a crucial role in many sensory and motor functions as well as in brain development, learning and memory and it is also involved in the pathogenesis of a number of neurological disorders, including epilepsy, Alzheimer's and Parkinson's diseases. Therefore, the study of Glu receptors (GluRs) is of therapeutical importance. We showed here by fluorescence monitoring of transmembrane Ca2+ ion fluxes in response to (S)-alpha-amino-3-hidroxi-5-metil-4-izoxazol propionic acid ((S)-AMPA) on the time scale of 0.00004-10 s that Ca2+ ion influx proceeds through faster and slower desensitizing receptors. Pharmacological isolation of the slower and faster desensitizing AMPA receptor was possible by fluorescence monitoring of Ca2+ ion translocation in response to (S)-AMPA in the presence and absence of various 2-methyl-4-oxo-3H-quinazoline-3-alkyl-carboxilic acid derivatives (Qxs): the acetic acid Q1 inhibits the slower desensitizing receptor response specifically, while the acetyl-piperidine Q5 is a more potent inhibitor of the faster desensitizing receptor response. In addition, spontaneous interictal activity, as induced by high [K+] conditions in hippocampal slices, was reduced significantly by Q5, suggesting a possible anticonvulsant property of Q5. Substitutions of Qxs into the GluR2 S1S2 binding core were consistent with their effect by causing variable degree of S1S2 bridging interaction as one of the main determinants of AMPA receptor agonist activity. The exploitation of differences between similar receptors will be important in the development and use of drugs with high pharmacological specificity.