Background and Purpose— The Na + /Ca 2+ exchanger, by mediating Ca 2+ and Na + fluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO). Methods— By means of Western blotting, NCX1, NCX2, and NCX3 protein expression was evaluated in the ischemic core and in the remaining nonischemic area of the slice at different time intervals starting from ischemia induction. The role of each isoform was also assessed with antisense oligodeoxynucleotides (ODNs) targeted for each isoform. These ODNs were continuously intracerebroventricularly infused with an osmotic minipump (1 μL/h) for 48 hours, 24 hours before pMCAO. Results— The results showed that after pMCAO all 3 NCX proteins were downregulated in ischemic core; NCX3 decreased in periinfarctual area whereas NCX1 and NCX2 were unchanged. The ODNs for NCX1 and NCX3 gene products were capable of inducing an increase in the ischemic lesion and to worsen neurological scores. Conclusions— The results of this study suggest that in the neuroprotective effect exerted by NCX during ischemic injury, the major role is prevalently exerted by NCX1 and NCX3 gene products.

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Background and Purpose-We characterized the differential effect of the NR2B subunit antagonist ifenprodil in the induction of activity-dependent long-term potentiation (LTP) and of postischemic LTP as well as in the neuronal damage induced by focal ischemia. Methods-Intracellular recordings were obtained from rat corticostriatal slice preparations. High-frequency stimulation of corticostriatal fibers was used as a LTP-inducing protocol. In vitro ischemia was induced by oxygen and glucose deprivation. In vivo ischemia was induced by permanent middle cerebral artery occlusion. Intracellular recordings were also performed in the ischemic penumbra. Results-Antagonists selectively targeting N-methyl-D-aspartate receptors containing the NR2B subunit blocked postischemic LTP without affecting activity-dependent LTP. In a model of focal ischemia, blockade of NR2B subunit in vivo caused reduction of brain damage, amelioration of neurological outcome, and normalization of the synaptic levels of NR2B subunits. Moreover, the antagonism of NR2B subunit was able to rescue the activity-dependent LTP in the ischemic penumbra. Conclusions-We suggest that NR2B subunits contribute to the striatal damage caused by in vivo and in vitro ischemia and play a critical role in the induction of postischemic LTP as well as in the suppression of activity-dependent LTP in the ischemic penumbra.

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Abstract:In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) occurred. Sodium nitroprusside (SNP), a nitric oxide donor and an iron-containing molecule, reduced chemical hypoxia-induced LDH release and cell death. These effects were counteracted by bepridil and by 5-(N-4-chlorobenzyl)-2Ј,4Ј-dimethylbenzamil (CB-DMB), two specific inhibitors of the Na ϩ -Ca 2ϩ exchanger. SNP also increased the activity of the Na ϩ -Ca 2ϩ exchanger as a Na ϩ efflux pathway, stimulated by Na ϩ -free conditions and evaluated by monitoring [Ca 2ϩ ] i in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca 2ϩ ] i elevation, and this effect was blocked by bepridil. Chemical hypoxiaevoked cell death and LDH release were counteracted by the ferricyanide moiety of the SNP molecule, K 3 Fe(CN) 6 , and by ferric chloride (FeCl 3 ), and this effect was counteracted by CB-DMB. In addition, the iron ion chelator deferoxamine reversed the protective effect exerted by SNP on cell injury. Collectively, these findings suggest that the protective effect of SNP on C6 glioma cells exposed to chemical hypoxia is due to the activation of the Na ϩ -Ca 2ϩ exchanger operating as a Na ϩ effluxCa 2ϩ influx pathway induced by iron present in the SNP molecule. Key Words: Na ϩ -Ca 2ϩ exchanger-Sodium nitroprusside -Iron-Chemical hypoxia-C6 gliomaCell survival. J. Neurochem. 74, 1505Neurochem. 74, -1513Neurochem. 74, (2000.The Na ϩ -Ca 2ϩ exchanger is a bidirectional pathway that couples the extrusion of Ca 2ϩ to the entrance of Na ϩ into the cell, or vice versa (Sanchez-Armass and Blaustein, 1987;Taglialatela et al., 1990). Under chemical hypoxia, a condition in which intracellular Ca 2ϩ and Na ϩ homeostasis is altered (Siesjö et al., 1989), the activation of the Na ϩ -Ca 2ϩ exchanger, when it is operating as a Na ϩ extrusion-Ca 2ϩ influx pathway, prevents cell damage in C6 glioma cells (Amoroso et al., 1997) and reduces aspartate release from the hippocampus (Amoroso et al., 1993). It has been recently reported that sodium nitroprusside (SNP), a nitric oxide (NO)-generating compound, possesses the ability to activate the Na ϩ -Ca 2ϩ exchanger in cultured rat astrocytes (Asano et al., 1995). On the other hand, the SNP molecule contains iron, besides the NO group. This metal ion, mediating the transfer of electrons between the cellular redox compounds and the appropriate disulfide-thiol groups of the Na ϩ -Ca 2ϩ exchanger molecule, can produce a stimulation of the antiporter activity (Reeves et al., 1986). Therefore, it appeared of interest to investigate (a) whether SNP could prevent chemical hypoxiainduced C6 glioma cell death, (b) whether this neuroprotective action was due to a stimulation of the Na ϩ -Ca 2ϩ exchanger activity, and (c) whether these effects were due to the NO donor property or to iron ions present in the SNP molecule.To this aim, C6 glioma cells, in which the activation o...

Over the last few years, although extensive studies have focused on the relevant function played by the sodium-calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na+]i and [Ca2+]i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.

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