Ischemic insults induce necroptotic cell death in hippocampal neurons through the up-regulation of endogenous RIP3.

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis, a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD), an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally, we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels, with a concomitant increase of the RIP1 protein. Together, these two related proteins form the necrosome, the complex responsible for induction of necroptotic cell death. Interestingly, we found that caspase-8 mRNA, a known negative regulator of necroptosis, was transiently decreased following OGD. Importantly, we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia, specifically in the CA1 area of the hippocampus. Moreover, we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together, our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury.

Excitotoxicity through Ca2+-permeable AMPA receptors requires Ca2+-dependent JNK activation.

The GluA4-containing Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (Ca-AMPARs) were previously shown to mediate excitotoxicity through mechanisms involving the activator protein-1 (AP-1), a c-Jun N-terminal kinase (JNK) substrate. To further investigate JNK involvement in excitotoxic pathways coupled to Ca-AMPARs we used HEK293 cells expressing GluA4-containing Ca-AMPARs (HEK-GluA4). Cell death induced by overstimulation of Ca-AMPARs was mediated, at least in part, by JNK. Importantly, JNK activation downstream of these receptors was dependent on the extracellular Ca(2+) concentration. In our quest for a molecular link between Ca-AMPARs and the JNK pathway we found that the JNK interacting protein-1 (JIP-1) interacts with the GluA4 subunit of AMPARs through the N-terminal domain. In vivo, the excitotoxin kainate promoted the association between GluA4 and JIP-1 in the rat hippocampus. Taken together, our results show that the JNK pathway is activated by Ca-AMPARs upon excitotoxic stimulation and suggest that JIP-1 may contribute to the propagation of the excitotoxic signal.

Regulation of AMPA receptors and synaptic plasticity.

Neuronal activity controls the strength of excitatory synapses by mechanisms that include changes in the postsynaptic responses mediated by AMPA receptors. These receptors account for most fast responses at excitatory synapses of the CNS, and their activity is regulated by various signaling pathways which control the electrophysiological properties of AMPA receptors and their interaction with numerous intracellular regulatory proteins. AMPA receptor phosphorylation/dephosphorylation and interaction with other proteins control their recycling and localization to defined postsynaptic sites, thereby regulating the strength of the synapse. This review focuses on recent advances in the understanding of the molecular mechanisms of regulation of AMPA receptors, and the implications in synaptic plasticity.

Role of the brain-derived neurotrophic factor at glutamatergic synapses.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays an important role in the activity-dependent regulation of synaptic structure and function, particularly of the glutamatergic synapses. BDNF may be released in the mature form, which activates preferentially TrkB receptors, or as proBDNF, which is coupled to the stimulation of the p75(NTR). In the mature form BDNF induces rapid effects on glutamate release, and may induce short- and long-term effects on the postsynaptic response to the neurotransmitter. BDNF may affect glutamate receptor activity by inducing the phosphorylation of the receptor subunits, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. Stimulation of the local protein synthesis and transcription activity account for the delayed effects of BDNF on glutamatergic synaptic strength. Several evidences show impaired synaptic plasticity of glutamatergic synapses in diseases where compromised BDNF function has been observed, such as Huntington's disease, depression, anxiety, and the BDNF polymorphism Val66Met, suggesting that upregulating BDNF-activated pathways may be therapeutically relevant. This review focuses on recent advances in the understanding of the regulation of the glutamatergic synapse by BDNF, and its implications in synaptic plasticity.

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

BDNF and Hippocampal Synaptic Plasticity.

Brain-derived neurotrophic factor (BDNF) belongs to a family of small secreted proteins that also include nerve growth factor, neurotrophin 3, and neurotrophin 4. BDNF stands out among all neurotrophins by its high expression levels in the brain and its potent effects at synapses. Several aspects of BDNF biology such as transcription, processing, and secretion are regulated by synaptic activity. Such observations prompted the suggestion that BDNF may regulate activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP), a sustained enhancement of excitatory synaptic efficacy thought to underlie learning and memory. Here, we will review the evidence pointing to a fundamental role of this neurotrophin in LTP, especially within the hippocampus. Prominent questions in the field, including the release and action sites of BDNF during LTP, as well as the signaling and molecular mechanisms involved, will also be addressed. The diverse effects of BDNF at excitatory synapses are determined by the activation of TrkB receptors and downstream signaling pathways, and the functions, typically opposing in nature, of its immature form (proBDNF). The activation of p75NTR receptors by proBDNF and the implications for long-term depression will also be addressed. Finally, we discuss the synergy between TrkB and glucocorticoid receptor signaling to determine cellular responses to stress.

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways.

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Contact sensitizers downregulate the expression of the chemokine receptors CCR6 and CXCR4 in a skin dendritic cell line.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response, namely in allergic contact dermatitis (ACD). In this work, we investigated by flow cytometry the effect of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulfate (NiSO(4)), on the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of skin DC. Our results show that all the skin sensitizers studied decreased the membrane expression of the chemokine receptors CCR6 and CXCR4. In contrast, 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB without contact sensitizing properties, was without effect on the surface expression of these receptors. Lipopolysaccharide (LPS), which induces the maturation of DC, also reduced surface CCR6 and CXCR4 expression.

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons.

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.

Synaptosomal [Ca2+]i as influenced by Na+/Ca2+ exchange and K+ depolarization.

The modulation of the intrasynaptosomal concentration of Ca2+, [Ca2+]i, by Na+/Ca2+ exchange was studied using Indo-1 fluorescence. The electrochemical gradient of Na+ was manipulated by substituting Li+ or choline for Na+ in the external medium and, then, the influx of 45Ca2+ and the [Ca2+]i were measured. It was found that the increase in [Ca2+]i induced by K+ depolarization is lower if the value of [Ca2+]i has been previously raised by Na+/Ca2+ exchange, suggesting that Ca2+ entering by Na+/Ca2+ exchange reduces the Ca2+ entering by voltage-dependent calcium channels. Our results show that a value of [Ca2+]i of about 650 nM induced by Na+/Ca2+ exchange reduces by 50% the Ca2+ entering due to K+ depolarization and no Ca2+ enters through the channels if the [Ca2+]i is previously raised above about 800 nM. Furthermore, predepolarization of the synaptosomes in a Ca-free medium also inhibits by at least 40% the [Ca2+]i rise through Ca2+ channels. Thus, the results suggest that both predepolarization and [Ca2+]i rise due to Na+/Ca2+ exchange decrease the Ca2+ entering by voltage-sensitive Ca2+ channels. The Ca2+ entering by Na+/Ca2+ exchange might contribute to the regulation of neurotransmitter release. Our results also show that the presence of Li+ in the external medium decreases the buffering capacity of synaptosomes, probably by releasing Ca2+ from mitochondria by Li+/Ca2+ exchange.

Non-specific effects of the MEK inhibitors PD098,059 and U0126 on glutamate release from hippocampal synaptosomes.

In order to investigate a role for the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) on hippocampal neurotransmitter release, we studied the effect of commonly used MEK (mitogen-activated protein kinase [MAPK]/ERK kinase) inhibitors, PD098,059 and U0126, on depolarization-induced glutamate release. PD098,059 inhibited glutamate release from hippocampal synaptosomes stimulated with 15 mM KCl in a concentration-dependent manner. At the same range of concentrations, PD098,059 inhibited basal and KCl-stimulated ERK1/2 phosphorylation. U0126, however, did not significantly affect KCl-evoked glutamate release at concentrations shown to inhibit ERK activity. Nonetheless, U0126 unspecifically potentiated depolarization-induced Ca2+-independent glutamate release, which masked a small dose-dependent inhibitory effect on the Ca2+-dependent release. PD098,059 reduced the [Ca2+]i response to KCl by partially inhibiting Ca2+ entry through N- and P-/Q-type voltage-gated Ca2+ channels, whereas U0126 did not affect depolarization-induced Ca2+ influx. To overcome the unspecific effect of PD098,059 on Ca2+ entry, we studied the effect of both MEK inhibitors on glutamate release stimulated by a Ca2+ ionophore. PD098,029 and U0126 showed a small dose-dependent inhibitory effect on ionomycin-induced glutamate release, at concentrations shown to inhibit ionomycin-stimulated ERK phosphorylation. These findings uncover new unspecific actions for both MEK inhibitors and suggest a minor role for ERK in modulating glutamate release in the hippocampus.

Nitric oxide differentially affects the exocytotic and the carrier-mediated release of [3H] gamma-aminobutyric acid in rat hippocampal synaptosomes.

We studied the effects of nitric oxide (NO) on the Ca2+-dependent KCl-evoked release of gamma-aminobutyric acid (GABA) by rat hippocampal synaptosomes, measured in the presence of 1-(2-(((diphenyl-methylene)amino)oxy)ethyl)-1,2,5, 6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), which blocks the GABA carrier. Under these conditions, the NO donor, hydroxylamine, up to 1 mM, inhibited the Ca2+-dependent exocytotic GABA release, but did not affect the basal release. However, in the absence of NNC-711, hydroxylamine concentrations higher than 30 microM caused a two-fold increase in the basal release of GABA, and the KCl-evoked release of GABA was higher than in the presence of NNC-711 because both exocytotic and carrier-mediated release occur. Thus, it is expected that when both release mechanisms are operative, NO inhibits the exocytotic release and stimulates the carrier-mediated release, and the overall effect is an increased liberation of the neurotransmitter from the nerve terminals.

Culture medium components modulate retina cell damage induced by glutamate, kainate or "chemical ischemia".

The aim of this study was to determine whether culture-conditioned medium (CCM) can prevent neuronal damage caused by excitotoxicity or by "chemical ischemia" in cultured chick retina cells. Excitotoxic conditions were obtained by incubating retina cells with glutamate or kainate and "chemical ischemia" was induced by metabolic inhibition. In this case, cultures were briefly exposed to sodium cyanide, to block oxidative phosphorylation and iodoacetic acid, to block glycolysis. The assessment of neuronal injury was made spectrophotometrically by quantification of cellularly reduced MTT. Stimulation of retina cells with glutamate or kainate in serum deprived culture medium (BME-FCS), lead to a decrease in the MTT metabolism that was dependent on the time of exposure to the toxic agents. CCM prevented cell damage, either when present during the stimulation period or during the recovery period. This protection was more prominent in the case of kainate-induced neuronal death. "Chemical ischemia" also lead to a decrease of the MTT metabolism in a time-dependent manner and CCM protected retina cells from "ischemia"-induced lesions when present during the stimulation period and during the recovery period. The protective effect of CCM was partially decreased by the tyrosine kinase inhibitor, genistein, when the cells were stimulated with kainate, but not with glutamate, or when the cells were subjected to "chemical ischemia". CCM protected retina cells against both the acute and the delayed toxicity induced by either glutamate or kainate, or by "chemical ischemia", when present during both the insult and the recovery period. The presence of survival factors in the media may effectively inhibit the cell death signals generated by glutamate receptor activation or by "chemical ischemia".

Domoic acid induced release of [3H]GABA in cultured chick retina cells.

The effect of the neurotoxin domoic acid (DOM), a structural analogue of kainic acid, on the release of [3H]gamma-aminobutyric acid (GABA) and on the [Ca2+]i was studied in cultured chick retina cells. DOM stimulated dose-dependently the release of [3H]GABA with an EC50 of 2.5 microM. In Ca(2+)-containing medium (1 mM), DOM (5 microM) increased the [Ca2+]i by about 190 nM and evoked the release of 11.8 +/- 1.3% of the intracellular [3H]GABA, while in the absence of extracellular Ca2+ DOM induced the release of only 7.9 +/- 1.4% of the accumulated [3H]GABA. The Ca(2+)-independent release of [3H]GABA was blocked by the non-competitive inhibitor of the GABA carrier 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-py ridine- carboxylic acid hydrochloride (NNC-711), but a component of Ca(2+)-dependent release remains. DOM evoked Ca(2+)-independent release of [3H]GABA was significantly depressed in the absence of external Na+ and completely blocked by the non-selective antagonist of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Similarly, CNQX decreased the [Ca2+]i response to DOM, whereas L(+)-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of the metabotropic glutamate receptors, was without effect. MK-801 did not affect the release of [3H]GABA stimulated by DOM. Taken together our results indicate that DOM evokes both Ca(2+)-dependent and Ca(2+)-independent release of [3H]GABA, most likely by activating kainate receptors.

Voltage-sensitive Ca2+ channels in rat striatal synaptosomes: role on the [Ca2+]i responses to membrane depolarization.

The fluorescent Ca2+ indicator Indo-1 was used to study the effect of depolarization evoked by KCl or 4-aminopyridine (4-AP) on the intracellular free calcium concentration responses (delta[Ca2+]i) in rat striatal synaptosomes. Depolarization of the synaptosomes with [KCl] > 7.5 mM induced a rapid increase of the [Ca2+]i followed by a decay towards a plateau. The size of the [Ca2+]i response varied sigmoidally with the synaptosomal membrane potential, with a transition potential of -27.3 mV. Depolarization with 4-AP evoked a dose-dependent sustained increase of the [Ca2+]i. Nitrendipine, omega-Conotoxin GVIA (omega-CgTx) and omega-Agatoxin IVA (omega-Aga IVA) were used to evaluate the relative role of L-, N-, P- and possibly Q-type voltage-sensitive Ca2+ channels (VSCCs) on the [Ca2+]i changes evoked by each of the two depolarizing agents. Nitrendipine caused only about 10% inhibition of the effect of either agent on the [Ca2+]i, suggesting that the L-type VSCCs have a modest contribution. The omega-CgTx decreased the response to KCl and 4-AP by 15 and 30%, respectively, but the latter effect may be partially due to a non-specific effect on Na+ channels. The omega-Aga IVA reduced the response to 4-AP by 26.5%, and this effect was additive to that of omega-CgTx, further suggesting that the striatal nerve terminals possess P- and/or Q-type, in addition to N-type Ca2+ channels. Neomycin (0.35 mM), tentatively used as an antagonist of the P-type channels, had a potent effect, decreasing the response to K(+)-depolarization and to 4-AP by, respectively, 32.5 and 48.5%. It is suggested that at the concentration used the antibiotic also partially blocks VSCCs which do not belong to the L-, N-, P- or Q-type VSCCs. We conclude that striatal nerve endings are equipped with at least four to five pharmacologically distinct classes of VSCCs, which are sensitive to well known antagonists of the L-, N-, P-, and Q-type VSCCs.

Kainate-induced retina amacrine-like cell damage is mediated by AMPA receptors.

We investigated the effect of domoate, kainate and AMPA on 45Ca2+ uptake and on metabolic activity of cultured chick amacrine-like cells, as measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Domoate and kainate stimulated 45Ca2+ uptake and decreased MTT reduction, in a LY 303070-sensitive manner. AMPA caused a small increase on 45Ca2+ uptake, but it was without effect on MTT reduction. AMPA reduced both the 45Ca2+ entry and neurotoxicity induced by kainate, and cyclothiazide enhanced both the 45Ca2+ entry and neurotoxicity induced by AMPA. The results indicate that the AMPA receptors are the non-NMDA glutamate receptors involved in excitotoxicity.

[3H]acetylcholine release from rat amacrine-like neurons is inhibited by adenosine A1 receptor activation.

We studied the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultures enriched (96.4+/-0.4%) in rat cholinergic amacrine-like neurons, as determined by labeling with an antibody against choline acetyltransferase. A small population of these cells also contained GABA. Using these cultures we observed that both [3H]ACh release, which was largely Ca2+-dependent, and 45Ca2+ influx, evoked by depolarization with 50 mM KCl, were increased when adenosine A1 receptor activation was prevented by removal of endogenous adenosine with adenosine deaminase, or by application of the A1 receptor antagonist DPCPX. Our results indicate that, in cultured rat amacrine-like neurons, the activation of A1 receptors decreases calcium influx and, thereby, inhibits [3H]ACh release.

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.

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.