Quinazolone-alkyl-carboxylic acid derivatives inhibit transmembrane Ca(2+) ion flux to (+)-(S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.

Comparison of the kinetics of the inward Ca(2+) ion flux to (S)-alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid [(S)-AMPA] in cerebrocortical homogenates and that of the previously reported transmembrane Na(+) ion influx mediated by an AMPA receptor in hippocampal homogenates established that the agonist-induced opening of the AMPA receptor channels occurs in two kinetically distinguishable phases. Here we report that the 2-methyl-4-oxo-3H-quinazoline-3-acetic acid (Q1) inhibits the major slow-phase response specifically, whereas the acetyl piperidine derivative (Q5) is a more potent inhibitor of the fast-phase response. Both the quinazolone-3-propionic acid (Q2) and the quinazolone-3-acetic acid methyl ester (Q3) enhanced the slow-phase response to (S)-AMPA. The information provided by docking different Q1, Q2, and Q5 models at the ligand-binding core of iGluRs were used to define agonistic and antagonistic modes of interactions. Based on the effects of quinazolone-3-alkyl-carboxylic acid derivatives on specific [(3)H]Glu binding and kinetically distinguishable Ca(2+) ion permeability responses to (S)-AMPA and molecular modeling, the fast- and the slow-phase (S)-AMPA-elicited Ca(2+) ion fluxes were corresponded to different subunit compositions and degrees of S1S2 bridging interaction relative to substitution of kainate thereupon. Substitutions of agonists and antagonists into the iGluR2 S1S2 ligand binding core induced different modes of domain-domain bridging.

Persistent depolarization and Glu uptake inhibition operate distinct osmoregulatory mechanisms in the mammalian brain.

The ways of coupling neuronal with glial compartments in natural physiology was investigated in microdialysis experiments by monitoring extracellular concentration of amino acids in the brain of anaesthetized rats. We hypothesized that extracellular [Glu], [Gln] and [Tau] patterns would be state-dependent. This was tested by stimulation of N-methyl-D-aspartate (NMDA) receptors, by inhibition of Glu uptake or by local depolarization with a high-K(+) dialysate, coupled with the addition of Co(2+) to block Ca(2+) influx. The results showed that (1) extracellular [Gln] was low whereas [Glu] and [Tau] were high during infusion of NMDA (0.5-1.0 mM) or high-K(+) (80 mM) in the hippocampus and ventrobasal thalamus, (2) hippocampal extracellular [Glu], [Gln] and [Tau] were increased in response to the Glu uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxilic acid (tPDC, 0.5-3.0 mM), in a concentration-dependent manner, (3) high-K(+)-induced increase of extracellular [Glu] was partially blocked by the addition of 10 mM CoCl(2) with the high-K(+) dialysate in the hippocampus. Searching for main correlations between changes in [Glu], [Gln] and [Tau] by calculating partial correlations and with the use of factor analyses we found, the primary response of the mammalian brain to persistent depolarization is the neuronal uptake of [Gln] and release of [Tau] thereupon, acting independently of Glu changes. When glial and neuronal uptake of Glu is blocked, releases of Tau occur from neuronal as well as glial compartments accompanied by increases of [Gln] in the mammalian brain.

Deramciclane inhibits N-methyl-D-aspartate receptor function.

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca(2+) ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 microM) significantly inhibited [(3)H]D-aspartate release and transmembrane Ca(2+) flux to N-methyl-D-aspartate in the absence of Mg(2+). By contrast, inhibition of [(3)H]D-aspartate release and transmembrane Ca(2+) flux evoked by 0.1 mM (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg(2+) and 10 microM cyclothiazide by 10 microM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 microM) did not inhibit [(3)H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.

Inhibition of [3H]D-aspartate release by deramciclane.

The effects of the 5-HT2C receptor inverse agonist deramciclane on the gamma-aminobutyric acid (GABA) uptake and excitatory amino acid release processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane non-competitively inhibited the uptake of [3H]GABA with a Ki value of 13.7 +/- 0.5 microM and partially displaced specifically bound [3H](R,S)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid ([3H]NNC-328) with high affinity (IC50 = 2.0 +/- 0.7 nM). Depolarization by 4-aminopyridine or by 4-aminopyridine with (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate [(S)-AMPA] induced the release of [3H]D-aspartate. Deramciclane (10 microM) partially (approximately 50%) inhibited the release of [3H]D-aspartate without affecting [3H]D-aspartate uptake. These results suggest a role for presynaptic inhibition of excitatory amino acid release and GABA uptake in the anxiolytic properties of deramciclane.

Role of intracellular Ca(2+) stores shaping normal activity in brain.

The role of intracellular Ca(2+) stores in the control of brain activity was investigated in microdialysis experiments by monitoring changes in the extracellular concentration of amino acids (AA) in the hippocampus of the rat after intracerebroventricular (icv) administration of the intracellular Ca(2+) release blocker, dantrolene in vivo, as well as in D-aspartate release and transmembrane Ca(2+) flux measurements in dantrolene-treated (50 microM) hippocampal homogenates containing resealed plasmalemma fragments and nerve endings in vitro. Microdialysis data demonstrate that icv injection of 0.6 mM dantrolene significantly decreases ( approximately 20%) the background (Glu) in the hippocampus. Both the (Glu; approximately 300%) and the inhibitory effect of dantrolene thereupon ( approximately 50%) was significantly increased when 0.5 mM of the Glu uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid, was dialysed into the hippocampus. NMDA and (S)-AMPA induced [(3)H]-D-aspartate release in hippocampal homogenates. Preincubation of these homogenates with 50 microM dantrolene was found to reduce the response to NMDA, but not to (S)-AMPA, in a NMDA-dependent manner. Increased rates of transmembrane influx and efflux of Ca(2+) in hippocampal homogenates with half-times of 4 ms and 200 ms, respectively, can be observed by the addition of 100 microM NMDA as recorded using a stopped-flow UV/fluorescence spectrometer in combination with the Ca(2+) indicator dye, bisfura-2. Both the Ca(2+) influx and efflux rates of the NMDA response were reduced (25-fold and >5-fold, respectively) in homogenates preloaded with 50 microM dantrolene. These results suggest a role for NMDA-inducible intracellular Ca(2+) stores in the control of normal brain activity in vivo.

Uridine activates fast transmembrane Ca2+ ion fluxes in rat brain homogenates.

The excitatory actions of the pyrimidine nucleoside uridine, and the nucleotides UDP and UTP, as well as the purine nucleotide ATP, were studied by fluorescent labeling of Ca2+ and K+ ion fluxes on the time scale of 0.04 ms to 10s in resealed plasmalemma fragments and nerve endings from the rat cerebral cortex. Two phases of Ca2+ ion influx with onsets of a few milliseconds and a few hundred milliseconds, showing different concentration dependencies, agonist sequences and subcellular localizations were distinguishable. [3H]Uridine identified high (K(D) approximately 15 nM) and low affinity (K(D)approximately 1 microM) specific binding sites in purified synaptosomal membranes. Labeled uridine taken up by synaptosomes in a dipyridamole-sensitive process was released by depolarization (1 mM 4-aminopyridine). Taken together, these results may qualify uridine as a neurotransmitter.

[Pyrimidine receptor function in the central nervous system]. / Pyrimidin receptor funkció a központi idegrendszerben.

A spectroscopic method, using fluorescent Ca2+, K+ and Na+ ion indicators in combination with the use of fast-kinetic techniques on the time scale of 0.00004-10 s has been applied to study mechanisms of P2 pyrimidoceptor-mediated signal transduction in brain homogenates. Effects of the known P2 receptor ligands (ATP, alpha, beta-methylene-ATP, UTP, UDP and uridine) and the P1 receptor ligand, adenosine, were compared by measuring the rates of transmembrane Ca2+, K+ and Na+ ion fluxes in resealed plasmalemma fragments and nerve endings from the rat cerebral cortex. In homogenates containing resealed plasmalemma fragments, uridine (0.03-30 microM), but not adenosine, activated two phases of Ca2+ ion influx with onsets of a few ms and hundred ms in a concentration-dependent manner. Also, the activation of the fast-phase Ca2+ ion response by ATP, UDP and alpha, beta-methylene-ATP whereas that of the slow-phase by UTP and UDP were observed with 3 microM concentration of these P2 receptor ligands. In homogenates containing resealed nerve endings, the fast-phase Ca2+ ion response to uridine was absent. UTP, but not uridine and UDP (3 microM), activated a fast K+ ion influx with onset of < 1 ms. Adenosine (3 microM) evoked a slow Na+ ion influx with onset of > 0.1 s whereas the influx of Na+ ion to uridine was detectable below 0.01 s. Both nucleotides, ATP and UTP (3 microM), activated fluctuations of transmembrane Na+ ion influx and efflux. By contrast, UDP caused efflux of Na+ ion in the subsecond range of time. Collectively these results suggest that transmembrane cation fluxes mediated by kinetically distinguishable P2U pyrimidoceptor subtypes are different.