Comparison of Effects of DL-Threo-β-Benzyloxyaspartate (DL-TBOA) and L-Trans-Pyrrolidine-2,4-Dicarboxylate (t-2,4-PDC) on Uptake and Release of [3H]D-Aspartate in Astrocytes and Glutamatergic Neurons

Uptake and release processes in cerebellar astrocytes and granule neurons (glutamatergic) for glutamate were investigated by the use of [³H]D-aspartate, a non-metabolizable glutamate analog. The effects of DL-threo--benzyloxyaspartate (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC) on uptake and release of [³H]D-aspartate were studied. Both compounds inhibited potently uptake of [³H]D-aspartate in neurons and astrocytes (IC₅₀ values 10-100 M), DL-TBOA being slightly more potent than t-2,4-PDC. Release of preloaded [³H]D-aspartate from neurons or astrocytes could be stimulated by addition of excess t-2,4-PDC whereas addition of DL-TBOA had no effect on [³H]D-aspartate efflux. Moreover, DL-TBOA inhibited significantly the depolarization-induced (55 mM KCl) release of preloaded [³H]D-aspartate in the neurons. The results reflect the fact that DL-TBOA is not transported by the glutamate carriers while t-2,4-PDC is a substrate which may heteroexchange with [³H]D-aspartate. It is suggested that DL-TBOA may be used to selectively inhibit depolarization coupled glutamate release mediated by reversal of the carriers.     

Selection of a pure cerebellar granule cell culture by kainate treatment

Chronic exposure of dissociated cerebellar cultures to 50M kainate results in a complete loss of [³H]-GABA release which is a marker of GABAergic interneurons. No loss of granule cells was found and the glutamatergic nature of the granule cells appeared unaltered by the kainate treatment, since evoked release of [³H]-d-aspartate was maintained after kainate exposure. Glial cells in such cultures are virtually eliminated by treatment with an antimitotic such as cytarabin. In consequence a pure culture of cerebellar granule cells virtually free of stellate, basket and glial cells may be obtained by a combined chronic treatment of the cultures with kainate and cytarabin.     

Selective effects of cyanide (100 μM) on the excitatory amino acid-induced elevation of intracellular calcium levels in neuronal culture

The effect of low concentrations of cyanide on the excitatory amino acid-induced elevations of intracellular calcium levels ([Ca²⁺]_i) was studied in cerebellar granule cells using ratio fluorometry with fura-2. Glutamate, kainate, N-methyl-d-aspartate (NMDA), quisqualate (50 M, each) and membrane depolarization by 40 mM KCl caused elevations of [Ca²⁺]_i which were 10-, 10-, 3-, 2.3-, 10-fold over baseline levels, respectively. Cyanide, 100 M, greatly augmented the increases in [Ca²⁺]_i induced by glutamate, kainate and NMDA but not those induced by quisqualate or KCl. In the absence of these excitatory amino acids, cyanide had no significant effect in concentrations up to 400 M. Elevations of [Ca²⁺]_i induced by quisqualate and KCl were not significantly augmented by higher concentrations of cyanide (400 M). Selective antagonists could block the effect of cyanide+the respective agonist; however, the calcium channel blockers, lanthanum and diltiazem lowered both NMDA- and kainate-induced elevations of [Ca²⁺]_i, yet neither blocked increases in calcium when 100 M cyanide was added. Collectively, these data support an interaction of cyanide with the excitatory amino acid receptor.     

Activation of adenosine A2 receptors enhances high K+-evoked taurine release from rat hippocampus: A microdialysis study

Summary The present study was designed to examine which type of adenosine receptors was involved in enhancement of high K⁺-evoked taurine release fromin vivo rat hippocampus using microdialysis. Perfusion with 0.5 or 5.0 mM adenosine enhanced high K⁺-evoked taurine release. Perfusion with 2M R(–)-N⁶-2-phenylisopropyladenosine (PIA), a selective adenosine A₁ receptor agonist, did not modulate taurine release. Perfusion with 1M 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A₁ receptor antagonist, increased taurine release. On the other hand, perfusion with 20M 2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethyl-carboxamideadenosine (CGS21680), a selective adenosine A_2A receptor agonist, enhanced taurine release, while perfusion with 1 mM 3,7-dimethyl-propagylxanthine (DMPX), an adenosine A₂ receptor antagonist, did not affect taurine release. These results demonstrate that adenosine enhances high K⁺-evoked taurine release via activation of adenosine A_2A receptors from both neurons and glial cells ofin vivo rat hippocampus.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.     

Inhibition of Glutamate-Induced Intensification of Free Radical Reactions by Gangliosides: Possible Role in Their Protective Effect in Rat Cerebellar Granule Cells and Brain Synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 M) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na⁺, K⁺-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Underestimation of primary production as indicated by measurements with size-fractionated phytoplankton in Lake Maarsseveen I (The Netherlands)

Primary productivity of four size classes of phytoplankton (<150m, <50m, <20m and <5m) was measured from March through October 1986 in Lake Maarsseveen I with an incubator technique. The mean column production was approximately 400 mg C.m^–2.day^–1, with a range of values between 150 and 750 mg C.m^–2.day^–1. The mean contribution of the size fractions <50m, <20m and <5m to the size fraction<150m was 80%, 60% and 35%, respectively. During their appearance the grazing impact of small herbivorous zooplankton,e.g. rotifers, can give an underestimation of the size fraction <150m. An indication of this phenomenon is given.     

The toxin helothermine affects potassium currents in newborn rat cerebellar granule cells

Helothermine, a recently isolated toxin from the venom of the Mexican beaded lizard Heloderma horridum horridum was tested on K⁺ currents of newborn rat cerebellar granule cells. In whole-cell voltageclamp experiments, cerebellar granule neurons exhibited at least two different K⁺ current components: a first transient component which is similar to an I _A-type current, is characterized by fast activating and inactivating kinetics and blocked by 4-aminopyridine; a second component which is characterized by noninactivating kinetics, is blocked by tetraetylammonium ions and resembles the classical delayed-rectifier current. When added to the standard external solution at concentrations ranging between 0.1 and 2 m helothermine reduced the pharmacologically isolated I _A-type current component in a voltage- and dose-dependent way, with a half-maximal inhibitory concentration (IC₅₀) of 0.52 m. A comparison between control and nelothermine-modified peak transient currents shows a slowdown of activation and inactivation kinetics. The delayed-rectifier component inhibition was concentration dependent (IC₅₀ = 0.86 m) but not voltage dependent. No frequency-or use-dependent block was observed on both K⁺ current types. Perfusing the cells with control solution resulted in quite a complete current recovery. We conclude that helothermine acts with different affinities on two types of K⁺ current present in central nervous system neurons.     

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[³H]glutamate. The apparent binding constant K _d was 135 nM and 393 nM and the maximal binding capacity B_max 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC₅₀ in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [³H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.     

Bilirubin Inhibits Ca2+-Dependent Release of Norepinephrine from Permeabilized Nerve Terminals

Although the well-known neurotoxic agent bilirubin can induce alterations in neuronal signaling, direct effects on neurotransmitter release have been difficult to demonstrate. In the present study we have used permeabilized nerve terminals (synaptosomes) from rat brain prelabeled with [³H]norepinephrine to examine the effects of bilirubin on transmitter release. Rat cerebrocortical synaptosomes were permeabilized with streptolysin-O (2 U/ml) in the absence or presence of bilirubin (10 M–320 M) and Ca²⁺ (100 M), and the amount of radiolabeled transmitter released during 5 min to the medium was analysed. Low levels of bilirubin decreased Ca²⁺-evoked release in a dose-dependent manner, with half-maximal effect at approx 25 M bilirubin. Higher levels of bilirubin (100–320 M) increased [³H]norepinephrine efflux in the absence of Ca²⁺, suggesting that high bilirubin levels induced leakage of transmitter from vesicles. The nontoxic precursor biliverdin had no effect on Ca²⁺-dependent exocytosis. Our data indicate that bilirubin directly inhibits both exocytotic release and vesicular storage of brain catecholamines.     

Effect of Subtype-Specific Ca2+-Antagonists and Ca2+-Free Media on the Field Stimulation-Evoked Release of ATP and [3H]Acetylcholine from Rat Habenula Slices

The involvement of different subtypes of voltage-sensitive Ca²⁺ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [³H]acetylcholine ([³H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [³H]ACh were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca²⁺ channel blocker -conotoxin GVIA (-CgTX, 0.01–1 M) reduced the stimulation-evoked release of ATP and [³H]ACh in a dose-dependent manner. Similarly, the P-type Ca²⁺ channel antagonist -agatoxin IVA (-Aga IVA) (0.05 M) and the inorganic Ca²⁺ channel blocker Cd²⁺ (0.2 mM) inhibited the outflow of both transmitters, while Ni²⁺ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca²⁺ antagonists. Long-term perfusion (i.e., 90 min) with Ca²⁺ free solution inhibited the evoked-release of ATP and [³H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [³H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca²⁺]₀ condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca²⁺ chelators, and dipyridamole (2 M), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca²⁺-free media. Tetrodotoxin (TTX, 1 M), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca²⁺]₀ conditions. In summary, we showed that both N- and P-type Ca²⁺ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [³H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [Ca²⁺]₀ conditions an additional release of ATP occurs, which is not associated with action potential propagation.     

Presynaptic modulation of amino acid release from synaptosomes

Using synaptosomes prepared from whole rat brain, the spontaneous, calcium-independent, and calcium-dependent release of glutamate and GABA was assessed. Time intervals of 1–30 seconds were studied. Spontaneous release of glutamate (but not GABA) was elevated by 10 M NMDA or AMPA by thirty seconds. This stimulation was partially calcium-dependent. Calcium-dependent release induced by 30 mM KCl was biphasic, confirming previous findings. This release was stimulated at all time periods by the presence of 10 M NMDA or AMPA in an antagonist-sensitive manner. These data suggest that glutamate and GABA are released from vesicular stores in rat synaptosomes and that some of this release is modulated by presynaptic glutamate receptors.     

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP₃), and on activation of protein kinase C (PKC) was studied in DDT₁ MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A₁ and some as yet unidentified P2Y receptor(s).2. Activation of either receptor type stimulated the production of InsP₃ and raised intracellular calcium in DDT₁ MF2 cells. Similarly, the A₁ selective agonist N⁶-cyclopentylade- nosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP_4–14 and induced a PKC translocation to the plasma membrane as determined using [³H]-phorbol dibutyrate (PDBu) binding in DDT₁ MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected -PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of -PKC-GFP and activation of PKC.3. Doses of the A₁ agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca²⁺ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 M) and UTP (or ATP, 2.5 M), which per se caused no detectable translocation of either - or -PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A₁ receptors synergistically increases Ca²⁺ transients and translocation of PKC in DDT₁ MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.     

Displacement of endogenous glutamate withd-aspartate: an effective strategy for reducing the calcium-independent component of glutamate release from synaptosomes

d-aspartate was used in the present study to partially deplete the cytosolic pool of glutamate, which is released independent of extracellular Ca²⁺, prior to measuring the K⁺-evoked release of this endogenous acidic amino acid from rat hippocampal mossy fiber synaptosomes. This pretreatment is known to be an effective method for substantially reducing the Ca²⁺-independent component of glutamate release. The rate of glutamate efflux is dependent on the concentration of sodium ions in the external medium and can be stimulated by exposure of hippocampal mossy fiber synaptosomes to externald-aspartate (50 M). Following the partial displacement of this cytosolic pool of glutamate withd-aspartate, the K⁺-evoked release of the residual, presumably vesicular, pool of endogenous glutamate has a strict requirement for external calcium and is highly dependent on the extent to which depolarization elevates the level of free cytosolic calcium. It is concluded that the protocol described in this study for the displacement of cytosolic glutamate withd-aspartate provides a useful alternative method of controlling for the Ca²⁺-independent component of glutamate release in synaptosomal preparations.Abbreviations used Ca calcium - Ca²⁺ free calcium - EGTA (ethylene-dioxy)diethylenedinitrilotetraacetic acid - KBM Krebs-bicarbonate medium The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

Haloperidol reduces K+-evoked Ca2+-dependentd-[3H]aspartate release from rat hippocampal slices

Rat hippocampal slices preloaded withd-[³H]aspartate, a non metabolizable analogue ofl-glutamate, were superfused with artifical CSF. Depolarization was induced by 53.5 mM K⁺, in the presence of Ca²⁺ (1.3 mM) or Mg²⁺ (5 mM) to determine the Ca²⁺ dependent release. Haloperidol added in the superfusion medium at 100 M reduced by about 60% the Ca²⁺ dependent release ofd-[³H]aspartate. This drug at 20 M or 100 M inhibited the non-activated glutamate dehydrogenase (GDH) but had no effect on GDH activated by ADP (2 mM) or leucine (5 mM). In addition no effect was observed on phosphate activated glutaminase (PAG) in the presence either of 20 mM or 5 mM phosphate. These results indicate that the effect of haloperidol is exerted on presynaptic mechanisms regulating neurotransmitter release.     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Differential alteration of dopamine,acetylcholine, and glutamate release during anoxia and/or 3,4-diaminopyridine treatment

The potassium-stimulated release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from mouse striatal slices was studied during anoxia and/or 3,4-diaminopyridine (DAP) treatment. Anoxia, in the presence of calcium, increased DA and GLU release, but depressed ACh release. Omission of calcium from an anoxic incubation further stimulated GLU and DA release and impaired ACh release. Under normoxic conditions, DAP (100 M) increased the release of all three neurotransmitters; the sensitivity of the slices to DAP changed with the presence or absence of an acetylcholinesterase inhibitor in the preincubation media. During an anoxic incubation, DAP did not ameliorate the anoxic-induced, K⁺-stimulated impairment of ACh release, but significantly reduced the K⁺-stimulated release of GLU and DA. These results are consistent with the hypothesis that hypoxia induces a presynaptic deficit that may underlie postsynaptic ischemic-induced changes. Amelioration of these presynaptic alterations in neurotransmitter release may be an effective approach to preventing hypoxic-induced damage.