Éva Szárics

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.

Effect of CGP 36742 on the extracellular level of neurotransmitter amino acids in the thalamus

We have evaluated the effect of the brain penetrating GABAb antagonist, CGP 36742 on GABAb receptors using in vivo microdialysis in the ventrobasal thalamus of freely moving rat. When a solution of 1 mM CGP 36742 in ACSF was dialyzed into the ventrobasal thalamus, 2-3-fold increases of extracellular Glu, Asp and Gly running parallel with significant decreases of contralateral extracellular Asp and Gly were observed. Unilateral applications of Glu receptor antagonists (0.5 mM MK801, 0.1 mM CNQX) evoked 2-3-fold decreases of CGP 36742-specific elevations of extracellular Asp, Glu and Gly. Administration of CNQX and MK801 in the absence of CGP 36742 did not alter the extracellular Glu and Gly concentrations whereas extracellular Asp concentrations diminished by 42-45% at both sides. By contrast, no changes of extracellular Gly accompanied the 5-10-fold enhancements of extracellular Asp and Glu, observed during application of the Glu uptake inhibitor, tPDC (1mM). Suspensions of resealed plasmalemma fragments from the rat thalamus were mixed rapidly with the membrane impermeant form of the fluorescence indicator, bis-fura-2 and the changes in fluorescence intensity in response to CGP 36742 (0.5 mM), and the GABAb agonist, baclofen (0.1 mM), were monitored on the time scale of 0.04 ms(-10)s. Progress of CGP 36742-mediated influx, and baclofen-mediated efflux of Ca++ ion, antagonized by CGP 36742, was observed in the 1 ms(-10s) period of time. These data support the hypothesis that background ventrobasal activities and thalamocortical signaling are under the control of inhibitory GABAb receptors in the ventrobasal thalamus.

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.

Kinetically distinguishable AMPA receptors in rat hippocampus are associated with the loss of glutamate-sensitive conformational transitions.

We describe a stopped-flow method to study alpha-amino-7-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-kainate receptor-mediated Na+ ion flux through native membranes. Resealed plasmalemma vesicles and nerve endings from the rat hippocampus were mixed rapidly with a membrane impermeant form of the fluorescence indicator, sodium binding benzofurane oxazole and the changes in fluorescence intensity in response to various [Glu] on the time scale of 0.04 ms-10 s were monitored at a sampling rate of 6.55 kHz. Inhibitors like ouabain (1 mM) and 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (dizocilpine, 50 microM) enhanced Na+ ion translocation under low-[Na+] and physiological conditions, respectively. Dependence of AMPA-kainate receptor kinetics on [Glu] was described in a model of channel activation by faster and slower desensitizing receptors. The model accounted for almost all of the Na+ ion flux activity in the 30 microM-10 mM range of [Glu]. We found that the values of the initial rate constant for Na+ ion influx, JA, and rate constant for desensitization, alpha, for the faster desensitizing receptor were dependent on data sampling rate, whereas the initial rate constant for Na+ ion flux through the slower desensitizing receptor, JB, varied much less with the sampling rate. These phenomena can be described by (1) a fractal model of short-lived AMPA-kainate receptor channel with many closely spaced states (fractal dimension approximately 1.8) and (2) a model of long-lived AMPA-kainate receptor channel with two discrete states.

Role of intracellular Ca2+ stores shaping normal activity in brain

The role of intracellular Ca2+ 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 Ca2+ release blocker, dantrolene in vivo, as well as in D‐aspartate release and transmembrane Ca2+ flux measurements in dantrolene‐treated (50 μM) hippocampal homogenates containing resealed plasmalemma fragments and nerve endings in vitro. Microdialysis data demonstrate that icv injection of 0.6 mM dantrolene significantly decreases (∼20%) the background (Glu) in the hippocampus. Both the (Glu; ∼300%) and the inhibitory effect of dantrolene thereupon (∼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 [3H]‐D‐aspartate release in hippocampal homogenates. Preincubation of these homogenates with 50 μM 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 Ca2+ in hippocampal homogenates with halftimes of 4 ms and 200 ms, respectively, can be observed by the addition of 100 μM NMDA as recorded using a stopped‐flow UV/fluorescence spectrometer in combination with the Ca2+ indicator dye, bisfura‐2. Both the Ca2+ influx and efflux rates of the NMDA response were reduced (25‐fold and >5‐fold, respectively) in homogenates preloaded with 50 μM dantrolene. These results suggest a role for NMDA‐inducible intracellular Ca2+ stores in the control of normal brain activity in vivo. J. Neurosci. Res. 57:906–915, 1999.

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.

Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter influx by Ca2+ in brain plasma membrane vesicles.

Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.

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.