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.

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 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.

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.

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.

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.