Carina Rodrigues Boeck

Ebselen prevents excitotoxicity provoked by glutamate in rat cerebellar granule neurons

Ebselen is a selenium compound that have glutathione peroxidase-like activity which is neuroprotective in acute stroke ischemia. The efficacy of ebselen to prevent excitotoxicity provoked by glutamate in cerebellar granule neurons was investigated at various time points and concentrations. Simultaneous addition of ebselen with glutamate decreased neuronal death and was completely reversed by 3 microM of ebselen (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide assays). However, when 1 microM of ebselen was added with glutamate and remained in the culture medium until 24 or 48 h, the neuronal survival increased to the control. The mechanism proposed for neuroprotection was the ability of ebselen to prevent lipoperoxidation provoked by glutamate. The present findings propose to amplify the use of ebselen in others neurodegenerative disorders involving glutamatergic system.

NMDA preconditioning protects against seizures and hippocampal neurotoxicity induced by quinolinic acid in mice.

Summary:u2002 Purpose: N‐methyl d‐aspartate (NMDA) preconditioning has been used to prevent cellular death induced by glutamate or NMDA in cultured neurons. Quinolinic acid (QA)‐induced seizures are used to average NMDA receptors–evoked neurotoxicity in animal models. The purpose of this study was to investigate the potential neuroprotective effects of NMDA preconditioning against QA‐induced seizures and hippocampal damage in vivo.

Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity.

N-Methyl-D-aspartate (NMDA) preconditioning is evoked by subtoxic concentrations of NMDA (50 microM), which has been shown previously to lead to transient resistance to subsequent lethal dose of glutamate or NMDA in cultured neurons. The purpose of this study was to investigate the participation of adenosine A1 and A2A receptors on NMDA preconditioning against glutamate-induced cellular damage in cerebellar granule cells. NMDA preconditioning prevented the stimulatory effect induced by glutamate on AMP hydrolysis, but not on ADP hydrolysis. The neuroprotection evoked by NMDA preconditioning against glutamate-induced cellular damage was prevented by the presence of adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 100 nM), but not by the adenosine A2A receptors antagonist, (4-(2[7-amino-2-(2-furyl {1,2,4}-triazolo{2,3-a{1,3,5}triazian-5-yl-aminoethyl)phenol (ZM 241385, 50 nM). Interestingly, a long-term treatment with CPT or ZM 241385 alone protected cells against glutamate-induced neurotoxicity. Moreover, the functionality of adenosine A1 receptor was not affected by NMDA preconditioning, but this treatment promoted adenosine A2A receptor desensitization, measured by cAMP accumulation. Taken together, the results described herein suggest that the neuroprotection evoked by NMDA preconditioning against cellular damage elicited by glutamate occurs through mechanisms involving adenosine A2A receptors desensitization co-operating with adenosine A1 receptors activation in cerebellar granule cells.

Phosphorylation of glial fibrillary acidic protein is stimulated by glutamate via NMDA receptors in cortical microslices and in mixed neuronal/glial cell cultures prepared from the cerebellum

In previous work we showed that phosphorylation of glial fibrillary acidic protein (GFAP), an astrocyte marker, is increased by glutamate in hippocampal slices from immature rats via a type II metabotropic receptor. In the present work we show that glutamate also stimulates GFAP phosphorylation in microslices prepared from immature cerebellar cortex, but by a different receptor mechanism from that observed in the hippocampus. Thus, in cerebellar microslices, NMDA consistently stimulated GFAP phosphorylation, whereas no effect of metabotropic or non-NMDA ionotropic agonists was observed. Glutamate and NMDA also stimulated GFAP phosphorylation in mixed neuronal/glial cell cultures from the cerebellum, although no effect of these agonists was observed in primary cultures of cerebellar astrocytes. In both models, the effects of glutamate and NMDA were dependent on external Ca(2+), were reversed by the NMDA receptor antagonist AP5 and were not blocked by tetrodotoxin. In the slice study the effect of NMDA was confined to a period starting with the first detectable expression of GFAP at 10 days and finishing at 16 days postnatal, as previously observed with metabotropic agonists in hippocampal slices. This period in the rat corresponds to the start of synaptogenesis when astrocyte hypertrophy is occurring. The results are discussed in the light of information in the literature on the occurrence of functional NMDA receptor subunits in glia.

Kinetic characterization and immunodetection of ecto-ATP diphosphohydrolase (EC 3.6.1.5) in cultured hippocampal neurons.

Extracellular ATP and adenosine modulate synaptic transmission in hippocampal neurons. ATP released from neural cells is hydrolyzed to adenosine by a chain of ecto-nucleotidases. ATP diphosphohydrolase hydrolyses ATP and ADP nucleotides to AMP and 5-nucleotidase hydrolyses AMP to adenosine. In this work, we investigated the ATPase and ADPase activities of ATP diphosphohydrolase in cultured hippocampal neurons. The apparent Michaelis-Menten constant (K(m)) was 233.9 +/- 14.6 and 221.8 +/- 63.6 microM, with a calculated maximal velocity (V(max), approximately) of 49.2 +/- 10.7 and 10.9 +/- 5.2 nmol Pi/mg protein/min for ATP and ADP, respectively. The horizontal straight line obtained in the competition plot indicated that only one active site is able to hydrolyze both substrates. Furthermore, we detected the presence of this enzyme using anti-CD39 antibody, which strongly stained the soma of pyramidal and bipolar neurons, but the neurites connecting the cell clusters were also immunopositive. This antibody recognized three bands with a molecular mass close to 95, 80 and 60kDa in immunoblotting analysis. The present results show, for the first time, the kinetic and immunocytochemical characterization of an ATP diphosphohydrolase in cultured hippocampal neurons. Probably, the widespread distribution of this enzyme on the surface of neurons in culture could reflect its functional importance in studies of synaptic plasticity hippocampal.

The modulation of ecto-nucleotidase activities by glutamate in cultured cerebellar granule cells

Several enzymes hydrolyze ATP, producing ADP which is hydrolyzed to AMP. Ecto-5′-nucleotidase produces adenosine from AMP. Glutamate (Glu) is an excitatory neurotransmitter and increases extracellular adenosine levels, which is considered an important inhibitory neuromodulator. Here we show that Glu activates ADP and AMP hydrolysis. NMDA and kainic acid (KA) also increased these enzymatic activities, but 1-aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD) had no effect. Dihydrokainate (DHK), an inhibitor of glutamate uptake, also blocked glutamate-evoked activation of ecto-nucleotidases, suggesting that this activation was also Glu transporters dependent. Therefore, we suggest that the Glu-evoked stimulation of ecto-nucleotidases might contribute to the increase of adenosine in extracellular space induced by Glu.

Effect of the L- or D-aspartate on ecto-5′nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A2A receptors

Summary.Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5′-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5′-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5′-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A2A receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A1 receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5′-nucleotidase and on cellular damage. The adenosine formed from ecto-5′-nucleotidase stimulation preferentially acted on adenosine A2A receptor which is probably co-operating with the neurotoxicity induced by amino acids.

Effects of glutamate transporter and receptor ligands on neuronal glutamate uptake

The excitatory amino acids (EAAs) transporters regulate the balance between physiological and pathological signaling over stimulation of the glutamatergic system pathway. The effect of transportable substrates and glutamate (Glu) receptor agonists on Glu uptake in neuronal cells was assessed at different conditions. Cells pre-incubated with Glu, L- or D-aspartate (Asp) and washed presented an inhibition on [(3)H]-Glu uptake and this effect was not mimicked by Glu receptors agonists. The effects of L- and D-Asp were not altered by the presence of N-methyl-d-aspartate (NMDA) receptor antagonists. Thus, the reduction on Glu uptake induced by EAAs is probably linked to the transporter activity. In contrast, the presence of NMDA or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) during the pre-incubation and the [(3)H]-Glu uptake assay period increased Glu uptake, whilst kainic acid (KA) had no effect. The NMDA effect was not altered by its antagonists (+/-)-2-amino-5-phosphonopentanoic acid (AP-5) or dizocilpine (MK-801). The SR-ACPD effect was due to the activation of metabotropic Glu receptor, since it was abolished by its antagonist, L(+/-)-2-amino-3-phosphonopropionic acid (L-AP3). Thus, the current studies suggest that the neuronal EAAs transporter is regulated in different manner by transportable substrates and Glu receptor agonists. The possible involvement of this modulation after certain neurotoxicity insults is discussed.