Spatial memory impairments in a prediabetic rat model.

Diabetes is associated with an increased risk for brain disorders, namely cognitive impairments associated with hippocampal dysfunction underlying diabetic encephalopathy. However, the impact of a prediabetic state on cognitive function is unknown. Therefore, we now investigated whether spatial learning and memory deficits and the underlying hippocampal dysfunction were already present in a prediabetic animal model. Adult Wistar rats drinking high-sucrose (HSu) diet (35% sucrose solution during 9 weeks) were compared to controls' drinking water. HSu rats exhibited fasting normoglycemia accompanied by hyperinsulinemia and hypertriglyceridemia in the fed state, and insulin resistance with impaired glucose tolerance confirming them as a prediabetic rodent model. HSu rats displayed a poorer performance in hippocampal-dependent short- and long-term spatial memory performance, assessed with the modified Y-maze and Morris water maze tasks, respectively; this was accompanied by a reduction of insulin receptor-ß density with normal levels of insulin receptor substrate-1 pSer636/639, and decreased hippocampal glucocorticoid receptor levels without changes of the plasma corticosterone levels. Importantly, HSu animals exhibited increased hippocampal levels of AMPA and NMDA receptor subunits GluA1 and GLUN1, respectively, whereas the levels of protein markers related to nerve terminals (synaptophysin) and oxidative stress/inflammation (HNE, RAGE, TNF-α) remained unaltered. These findings indicate that 9 weeks of sucrose consumption resulted in a metabolic condition suggestive of a prediabetic state, which translated into short- and long-term spatial memory deficits accompanied by alterations in hippocampal glutamatergic neurotransmission and abnormal glucocorticoid signaling.

Functional interaction between pre-synaptic α6ß2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum.

BACKGROUND AND PURPOSE: Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A2A receptors (A2A Rs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson's disease and addiction. Since A2A Rs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A2A Rs and nAChRs in rat striatal dopaminergic terminals. EXPERIMENTAL APPROACH: We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo. KEY RESULTS: In striatal nerve terminals, the selective A2A R agonist CGS21680 inhibited, while the A2A R antagonist ZM241385 potentiated the nicotine-stimulated [(3) H]dopamine ([(3) H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [(3) H]DA release was no longer modulated by A2A R ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal. CONCLUSIONS AND IMPLICATIONS: These results provide the first evidence for a functional interaction between nicotinic and adenosine A2A R in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson's disease and other dopaminergic disorders.

Cell cycle re-entry in Alzheimer's disease: a major neuropathological characteristic?

Alzheimer's disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and thinking skills. With over 26 million patients in 2006, AD is the most prevalent neurodegenerative disease worldwide. Different hypotheses have been suggested to explain the pathogenesis of AD, like those involving inflammation, mitochondrial dysfunction or oxidative stress. Many of these studies have addressed amyloid plaque formation, tau hyperphosphorylation and apoptotic neuronal loss, the three main histopathological hallmarks of this disease. Increasing evidences, however, suggest another feature that can also be considered a neuropathological marker for AD: ectopic cell cycle re-entry. Cell cycle events have been frequently registered in the brains of AD patients. Although the exact starting point of cell cycle re-entry remains unclear, a number of subsequent cascades, which include events such as kinase upregulation, DNA replication and cytoskeletal alterations, have already been described. There are also increasing reports suggesting that cell cycle reactivation in mature neurons occurs as part of the apoptotic process. Upon a brief overview of the different theories and models addressing cell cycle reactivation in AD, we will describe possible mechanisms that trigger cell cycle re-entry, with special attention to links between this feature and the main neuropathological markers of AD. Finally, we will also analyze possible similarities between cell cycle dysregulation in AD and in other pathologies, such as Prion-Related Encephalopathies.

Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: involvement of oxidative stress and mitogen-activated protein kinase cascades.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder primarily characterized by excessive deposition of amyloid-beta (Abeta) peptides in the brain. One of the earliest neuropathological changes in AD is the presence of a high number of reactive astrocytes at sites of Abeta deposition. Disturbance of glutamatergic neurotransmission and consequent excitotoxicity is also believed as implicated in the progression of this dementia. Therefore, the study of astrocyte responses to Abeta, the main cellular type involved in the maintenance of synaptic glutamate concentrations, is crucial for understanding the pathogenesis of AD. This study aims to investigate the effect of Abeta on the astrocytic glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST), and their relative participation to glutamate clearance. In addition we have also investigated the involvement of mitogen-activated protein (MAP) kinases in the modulation of GLT-1 and GLAST levels and activity and the putative contribution of oxidative stress induced by Abeta to the astrocytic glutamate transport function. Therefore, we used primary cultures of rat brain astrocytes exposed to Abeta synthetic peptides. The data obtained show that Abeta(1-40) peptide decreased astroglial glutamate uptake capacity in a non-competitive mode of inhibition, assessed in terms of tritium radiolabeled d-aspartate (d-[(3)H]aspartate) transport. The activity of GLT-1 seemed to be more affected than that of GLAST, and the levels of both transporters were decreased in Abeta(1-40)-treated astrocytes. We demonstrated that MAP kinases, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase, were activated in an early phase of Abeta(1-40) treatment and the whole pathways differentially modulated the glutamate transporters activity/levels. Moreover it was shown that oxidative stress induced by Abeta(1-40) may lead to the glutamate uptake impairment observed. Taken together, our results suggest that Abeta peptide downregulates the astrocytic glutamate uptake capacity and this effect may be in part mediated by oxidative stress and the differential activity and complex balance between the MAP kinase signaling pathways.

Susceptibility of hippocampal neurons to Abeta peptide toxicity is associated with perturbation of Ca2+ homeostasis.

Neuritic dystrophy, loss of synapses and neuronal death in the cerebral cortex and hippocampus are hallmarks of Alzheimer's disease. The aim of the present study was to investigate the differential susceptibility of cortical and hippocampal neurons to amyloid-beta (Abeta)-induced toxicity. For that, we have used primary neuronal cultures prepared from rat brain cortex and hippocampus which were treated with the synthetic peptides Abeta25-35 or Abeta1-40. Abeta-induced apoptotic cell death was analyzed by determining caspase-3-like activity. Neuritic dystrophy was evaluated by cobalt staining and MAP2 immunoreactivity. Perturbation of Ca(2+) homeostasis caused by exposure to Abeta was evaluated by determining basal cytosolic calcium levels in the whole neuronal population and by single cell calcium imaging under basal and KCl-depolarization conditions. Finally, levels of GluR2 subunit of glutamate AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate) receptors were quantified by western blotting. Our results demonstrated that hippocampal neurons in culture are more susceptible than cortical neurons to Abeta-induced apoptosis and also that this mechanism involves the perturbation of Ca(2+) homeostasis. Accordingly, the exposure of hippocampal neurons to Abeta peptides decreases the protein levels of the GluR2 subunit of glutamate AMPA receptors that may be associated with a significant rise of cytosolic Ca(2+) concentration, leading to dendritic dystrophy and activation of apoptotic neuronal death.

Alzheimer's disease-associated neurotoxic mechanisms and neuroprotective strategies.

The characteristic hallmarks of Alzheimer's disease (AD), the most common form of dementia in the elderly, include senile plaques, mainly composed of beta-amyloid (Abeta) peptide, neurofibrillary tangles and selective synaptic and neuronal loss in brain regions involved in learning and memory. Genetic studies, together with the demonstration of Abeta neurotoxicity, led to the development of the amyloid cascade hypothesis to explain the AD-associated neurodegenerative process. However, a modified version of this hypothesis has emerged, the Abeta cascade hypothesis, which takes into account the fact that soluble oligomeric forms and protofibrils of Abeta and its intraneuronal accumulation also play a key role in the pathogenesis of the disease. Recent evidence posit that synaptic dysfunction triggered by non fibrillar Abeta species is an early event involved in memory decline in AD. The current understanding of the molecular mechanisms responsible for impaired synaptic function and cognitive deficits is outlined in this review, focusing on oxidative stress and disturbed metal ion homeostasis, Ca(2+) dysregulation, mitochondria and endoplasmic reticulum dysfunction, cholesterol dyshomeostasis and impaired neurotransmission. The activation of apoptotic cell death as a mechanism of neuronal loss in AD, and the prominent role of neuroinflammation in this neurodegenerative disorder, are also reviewed herein. Furthermore, we will focus on the more relevant therapeutical strategies currently used, namely those involving antioxidants, drugs for neurotransmission improvement, hormonal replacement, gamma- and beta- secretase inhibitors, Abeta clearance agents (Abeta immunization, disruption of Abeta fibrils, modulation of the cholesterol-mediated Abeta transport), non-steroidal anti-inflammatory drugs (NSAIDs), microtubules stabilizing drugs and kinase inhibitors.

Neurodegenerative pathways in Parkinson's disease: therapeutic strategies.

Parkinson's disease (PD), considered one of the major neurological disorders, is characterized by the loss of dopaminergic neurons in the pars compacta of the substantia nigra and by the presence of intraneuronal cytoplasmic inclusions called Lewy bodies. The causes for degeneration of PD neurons remain unclear, however, recent findings contributed to clarify this issue. This review will discuss the current understanding of the mechanisms underlying Parkinson's disease pathogenesis, focusing on the current and potential therapeutic strategies for human treatment.

Adenosine A2A receptors regulate the extracellular accumulation of excitatory amino acids upon metabolic dysfunction in chick cultured retinal cells.

The role of endogenous extracellular adenosine as a tonic modulator of the extracellular accumulation of excitatory amino acids (glutamate and aspartate) caused by metabolic inhibition was investigated in cultured retinal cells. The selective adenosine A2A receptor antagonist, 4-[2-[7-amino-2-(2-furyl)(1,2,4)-triazin-5-ylamino]-ethyl]ph enol (ZM241385) (50 nM), increased the release of glutamate (three- to four-fold) and of aspartate (nearly two-fold) upon iodoacetic acid-induced glycolysis inhibition, in the presence or in the absence of Ca2+. Blockade of tonic activation of A2A receptors by ZM241385 also increased (nearly two-fold) the ischemia-induced release of glutamate and aspartate. Furthermore, another selective A2A receptor antagonist, 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5- c] pyrimidine (SCH58261), also increased the release of aspartate and glutamate by about two-fold in cells submitted to glycolysis inhibition. In contrast, the selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (100 nM), did not significantly modify the extracellular accumulation of either glutamate or aspartate caused by inducers of chemical ischemia or glycolytic inhibitors. Inhibition of glycolysis also increased (about three-fold) the extracellular accumulation of GABA, which was virtually unchanged by ZM241385. Furthermore, the GABAA receptor antagonist, bicuculline (10 microM), only increased (nearly two-fold) the iodoacetic acid-induced Ca(2+)-dependent release of glutamate, whereas the GABAB receptor antagonist, 3-aminopropyl(diethoxymethyl) phosphinic acid, CGP35348 (100 microM), was devoid of effects on the extracellular accumulation of glutamate and aspartate. These results show that endogenous extracellular adenosine, which rises under conditions of inhibited glycolysis, tonically inhibits the extracellular accumulation of excitatory amino acid through the activation of A2A, but not A1, adenosine receptors, and this effect is independent of GABAA and GABAB functions in the cultured retinal cells.

Adenosine modulation of D-[3H]aspartate release in cultured retina cells exposed to oxidative stress.

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.

Vinpocetine attenuates the metabolic dysfunction induced by amyloid beta-peptides in PC12 cells.

The cytoprotective effect of vinpocetine [14-ethoxycarbonyl-(3alpha, 16alpha-ethyl)-14,15-eburnamine] was investigated on PC12 cells treated with the amyloid beta-peptides (Abeta) for 24 hours. Vinpocetine was shown to protect cells from the inhibition in redox status induced by exposure to Abeta25-35 and Abeta1-40, the maximal protection being achieved at a vinpocetine concentration of 40 microM. At this concentration, vinpocetine blocked the inhibition of the mitochondrial respiratory chain complexes II-III and IV and completely abolished the depletion of pyruvate levels induced by toxic concentrations of Abeta peptides. Furthermore, the accumulation of ROS in cells exposed to Abeta25-35 and Abeta1-40 evaluated using the fluorescent probe 2',7'-dichlorofluorescin (DCF), was reduced in the presence of 40 microM vinpocetine. Taken together, the data presented herein demonstrate that vinpocetine protects cells from Abeta toxicity, preventing the generation of oxidative stress due to the excessive accumulation of ROS. This study suggests that vinpocetine can exert neuroprotective properties which might be of importance and contribute to its clinical efficacy in the treatment of Alzheimer's disease or other neurodegenerative disorders in which oxidative stress is involved.

Glutamate in life and death of retinal amacrine cells.

1. Glutamate is the neurotransmitter released by bipolar cells at their synapses with amacrine cells. The amacrine cells express ionotropic (NMDA, AMPA and kainate) and metabotropic (mGluR1, mGluR2, mGluR4 and mGluR7) glutamate receptors and may take up glutamate from the synaptic cleft. 2. Activation of the ionotropic glutamate receptors increases the intracellular free calcium concentration ([Ca2+]i), owing to Ca2+ entry through the receptor-associated channels as well as through voltage-gated Ca2+ channels. The [Ca2+]i response to glutamate may be amplified by Ca2+-induced Ca2+ release from intracellular sources. 3. Activation of NMDA and non-NMDA glutamate receptors stimulates the release of GABA and acetylcholine from amacrine cells. GABA is released by a Ca2+-dependent mechanism and by reversal of the neurotransmitter transporter. 4. Excessive activation of glutamate receptors during ischemia leads to amacrine cell death. An increase in [Ca2+]i due to Ca2+ influx through NMDA and AMPA/kainate receptor channels is related to cell death in studies in vitro. In other studies, it was shown that nitric oxide may also take part in the process of cell damage during ischemia.

Oxidative stress affects the selective ion permeability of voltage-sensitive Ca2+ channels in cultured retinal cells.

The effect of ascorbate/Fe2+-induced oxidative stress on the intracellular Ca2+ concentration ([Ca2+]i) and on the voltage-sensitive Ca2+ channels (VSCC) of chick retinal cells was evaluated in this study. We also analyzed the effect of oxidation on the intracellular Na+ concentration ([Na+]i) and on the Ca2+-dependent release of [3H])gamma-aminobutric acid (GABA) evoked by 50 mM KCI. The resting [Ca2+]i was not affected by oxidation, but the [Ca2+]i response (delta[Ca2+]i) to K+-depolarization was significantly inhibited under oxidative stress conditions. The Ca2+ influx stimulated by membrane depolarization was mediated by L- and N-type VSCC, and by N-metyl-D-aspartate (NMDA) receptor channel, activated by endogenous glutamate released by glutamatergic cells. In cultured retinal cells L-type channels are the major route of Ca2+ influx during depolarization and the most affected by oxidative stress. The N-type VSCC seem not to be affected by oxidant conditions; they were found to be involved in glutamatergic transmission and only indirectly in the release of [3H]GABA evoked by K+-depolarization. Although the Ca2+-dependent release of [3H]GABA evoked by 50 mM KCl is mediated by Ca2+ entry through L-type Ca2+ channels, it is not affected by pre-incubation with the oxidant pair. The oxidative stress conditions increased the [Na+]i in Ca2+-free medium, by a process dependent of Na+ entry through L-type VSCC. The increased permeability of L-type VSCC to Na+ may increase the Ca2+-independent release of endogenous glutamate which, by activating the NMDA receptors, induces the release of [3H]GABA by reversal of its transporter. The equilibrium between the release of GABA and glutamate may play an in important role in neuroprotection against excitotoxic insults.

Impairment of excitatory amino acid transporter activity by oxidative stress conditions in retinal cells: effect of antioxidants.

In the present study we analyzed how oxidative stress conditions induced by ascorbate/ Fe2+ affect the excitatory amino acid (EAA) transport systems in cultured chick retina cells. The uptake of D-[3H]aspartate, which is transported by the same carrier as glutamate, was determined in control cells and in cells subjected to ascorbate/Fe2+. The uptake of this EAA was Na+ dependent and was inhibited by about 40% under oxidative stress conditions. To clarify the molecular mechanisms involved in the inhibition of D-[3H]aspartate uptake by ascorbate/Fe2+, we investigated the effect of vitamin E (Vit E), melatonin, reduced glutathione (GSH), and dithiothreitol (DTT) on the uptake of D-[3H]aspartate and on the extent of lipid peroxidation in control and in peroxidized cells. Preincubation with Vit E (100 microM) abolished lipid peroxidation, but had no significant effect on the inhibition of D-[3H]aspartate uptake evoked by ascorbate/Fe2+. Melatonin was more effective in reducing the formation of TBARS and conjugated dienes than in preventing the D-[3H]aspartate uptake inhibition evoked by the oxidant pair. Conversely, GSH (4 mM) and DTT (4 mM) completely prevented the inhibition of D-[3H]aspartate uptake in cells subjected to oxidative stress, but were without effect on the extent of peroxidation. Free fatty acids, such as arachidonic acid, seem not to be involved in reducing the activity of the D-[3H]aspartate uptake system, whereas the reduction of the Na+ electrochemical gradient that occurs under oxidative stress was in part involved in the reduction of D-[3H]aspartate uptake by the cells. The inhibition of D-[3H]aspartate uptake by ascorbate/Fe2+ persisted for at least 1 h, but could be partially reverted by disulfide reducing agents. It is concluded that oxidative stress causes long-lasting modifications of the glutamate/D-[3H]aspartate transport system (or systems), such as oxidation of protein sulfhydryl (SH) groups, which can be recovered by some antioxidants.

Activity of ionotropic glutamate receptors in retinal cells: effect of ascorbate/Fe(2+)-induced oxidative stress.

The effect of oxidative stress induced by the oxidant pair ascorbate/Fe2+ on the activity of ionotropic glutamate receptors was studied in cultured chick retina cells. The release of [3H]GABA and the increase of the intracellular free Na+ concentration ([Na+]i), evoked by glutamate receptor agonists, were used as functional assays for the activity of the receptors. The results show that the maximal release of [3H]GABA evoked by kainate (KA; approximately 20% of the total) or AMPA (approximately 11% of the total) was not different in control and peroxidized cells, whereas the EC50 values determined for peroxidized cells (33.6 +/- 1.7 and 8.0 +/- 2.0 microM for KA and AMPA, respectively) were significantly lower than those determined under control conditions (54.1 +/- 6.6 and 13.0 +/- 2.2 microM for KA and AMPA, respectively). The maximal release of [3H]GABA evoked by NMDA under K+ depolarization was significantly higher in peroxidized cells (7.5 +/- 0.5% of the total) as compared with control cells (4.0 +/- 0.2% of the total), and the effect of oxidative stress was significantly reduced by a phospholipase A2 inhibitor or by fatty acid-free bovine serum albumin. The change in the intracellular [Na+]i evoked by saturating concentrations of NMDA under depolarizing conditions was significantly higher in peroxidized cells (8.9 +/- 0.6 mM) than in control cells (5.9 +/- 1.0 mM). KA, used at a subsaturating concentration (35 microM), evoked significantly greater increases of the [Na+]i in peroxidized cells (11.8 +/- 1.7 mM) than in control cells (7.1 +/- 0.8 mM). A saturating concentration (150 microM) of this agonist triggered similar increases of the [Na+]i in control and peroxidized cells. Accordingly, the maximal number of binding sites for (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([3H]MK-801) was increased after peroxidation, whereas the maximal number of binding sites for [3H]KA was not affected by oxidative stress. These data suggest that under oxidative stress the activity of the ionotropic glutamate receptors is increased, with the NMDA receptor being the most affected by peroxidation.

Intracellular free Na+ concentration increases in cultured retinal cells under oxidative stress conditions.

The effect of oxidative stress, induced by ascorbate/Fe2+, on the intracellular free Na+ concentration ([Na+]i) of cultured chick retina cells was determined using the fluorescent indicator Na(+)-binding benzofuran isophthalate (SBFI). The resting[Na+]i of retina cells submitted to oxidative stress (15.5 +/- 1.9 mM) was significantly higher than that of control cells (8.9 +/- 0.8 mM). KCl (50 mM) depolarization induced a sustained [Na+]i increase (delta[Na+]i), which was significantly higher in peroxidized cells (8.1 +/- 0.7 mM) than in control cells (4.9 +/- 0.9 mM). The glutamate receptor antagonists, MK-801 and CNQX, reduced more significantly the initial delta[Na+]i induced by K(+)-depolarization under oxidative stress conditions (65% of inhibition), than in control cells (20% of inhibition). Moreover, in the presence of MK-801 and CNQX the increase in the [Na+]i, which was similar in control and peroxidized cells, was followed by a decrease towards a plateau. The Na+ channel blocker, tetrodotoxin (TTX), also reduced the sustained increase of the [Na+]i evoked by 50 mM KCl in both experimental conditions. However, TTX and glutamate receptor antagonists tested together failed to abolish the delta[Na+]i upon K(+)-depolarization, indicating that TTX-resistant Na+ channels were involved in the Na+ influx. The entry of Na+ through these channels contributed mainly to the early phase of the [Na+]i rise upon K(+)-depolarization, whereas the glutamate receptors seem to contribute more significantly to the [Na+]i response for stimulations longer than 30-50 s. The results suggest that an excessive activation of glutamate receptors increases the influx of Na+ and the resting [Na+]i under oxidative stress conditions.

Modulation of N-methyl-D-aspartate receptor activity by oxidative stress conditions in chick retinal cells.

The effect of oxidative stress, induced by ascorbate (1.5 mM)/Fe2+ (7.5 microM), on the cellular responses to N-methyl-D-aspartate (NMDA) receptor activation was evaluated by measuring the release of [3H]GABA induced by NMDA from cultured retina cells. In retina cells submitted to oxidative stress the [3H]GABA release evoked by NMDA, in a medium containing physiological concentrations of Mg2+ (1.6 mM) and K+ (4 mM), was significantly higher than in control cells. The [3H]GABA release evoked by NMDA was potentiated by glycine and was abolished by MK-801, suggesting that the [3H]GABA release was due to NMDA receptor activation. The increased effect of NMDA in peroxidized cells was significantly reduced by TTX, suggesting that the higher cellular responses to the activation of NMDA receptors are due to a hyperexcitability of retina cells submitted to oxidative stress. No significant differences were found between the average resting membrane potential of control and peroxidized cells. However, membrane potential is more tightly regulated by K(+)-channels sensitive to 4-aminopyridine (100 microM), alpha-dendrotoxin (100 nM) and gamma-dendrotoxin (100 nM) under oxidative stress.

Survival of allochthonous bacteria in still mineral water bottled in polyvinyl chloride (PVC) and glass.

The mortality of Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa, based on the culturability of these bacteria, was assessed in non-carbonated mineral water, bottled in polyvinyl chloride (PVC) containing the indigenous flora, sterile mineral water bottled in PVC, sterile mineral water in glass containers, and sterile tap water in glass containers. There was a general decrease in the culturability of these organisms in the four test waters, except that Ps. aeruginosa grew in sterile tap water. Escherichia coli and Kl. pneumoniae had the highest mortality rates under the conditions tested, while Ent. cloacae had a very low and constant mortality rate that would have resulted in the persistence of this organism in mineral water for a long period of time. After a sharp initial decrease in culturability, Ps. aeruginosa also had a very low mortality rate in mineral water bottled in PVC.

Effect of oxidative stress on the release of [3H]GABA in cultured chick retina cells.

The effect of ascorbate (1.5 mM)/Fe2+ (7.5 microM)-induced oxidative stress on the release of pre-accumulated [3H]gamma-aminobutyric acid ([3H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K+ increased the release of [3H]GABA by 1.01 +/- 0.16% and 2.5 +/- 0.3% of the total, in the absence and in the presence of Ca2+, respectively. Lipid peroxidation increased the release of [3H]GABA to 2.07 +/- 0.31% and 3.6 +/- 0.39% of the total, in Ca(2+)-free or in Ca(2+)-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-py ridine- carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [3H]GABA due to K(+)-depolarization in the absence of Ca2+, but only 65% of the release occurring in the presence of Ca2+ in control and peroxidized cells. Under oxidative stress retina cells release more [3H]GABA than control cells, being the Ca(2+)-independent mechanism, mediated by the reversal of the Na+/GABA carrier, the most affected. MK-801 (1 microM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [3H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 48%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [3H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 microM) stimulated [3H]GABA release evoked by 50 mM K+-depolarization in control but not in peroxidized cells.(ABSTRACT TRUNCATED AT 250 WORDS)