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Naϩ /Ca 2ϩ exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na ϩ and Ca 2ϩ homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3Ϫ/Ϫ mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3Ϫ/Ϫ mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3Ϫ/Ϫ mice exposed to OGD plus reoxygenation. In addition, in ncx3Ϫ/Ϫ cortical neurons exposed to hypoxia, NCX currents, recorded in the reverse mode of operation, were significantly lower than those detected in ncx3ϩ/ϩ. From these results, NCX3 protein emerges as a new molecular target that may have a potential therapeutic value in modulating cerebral ischemia.

In the current study, the potential blocking ability of K+ channels encoded by the human ether-a-go-go related gene (HERG) by the piperazine H1 receptor antagonist cetirizine has been examined and compared with that of other second-generation antihistamines (astemizole, terfenadine, and loratadine). Cetirizine was completely devoid of any inhibitory action on HERG K+ channels heterologously expressed in Xenopus laevis oocytes in concentrations up to 30 microM. On the other hand, terfenadine and astemizole effectively blocked HERG K+ channels with nanomolar affinities (the estimated IC50 values were 330 and 480 nM, respectively), whereas loratadine was approximately 300-fold less potent (IC50 approximately 100 microM). In addition, in contrast to terfenadine, cetirizine did not show use-dependent blockade. In SH-SY5Y cells, a human neuroblastoma clone that constitutively expresses K+ currents carried by HERG channels (IHERG), as well as in human embryonic kidney 293 cells stably transfected with HERG cDNA, extracellular perfusion with 3 microM cetirizine did not exert any inhibitory action on IHERG. Astemizole (3 microM), on the other hand, was highly effective. Terfenadine (3 microM) caused a marked (approximately 80%) inhibition of IHERG in SH-SY5Y cells, whereas loratadine, at the same concentration, caused a 40% blockade. Furthermore, the application of cetirizine (3 microM) on the intracellular side of the membrane of HERG-transfected human embryonic kidney 293 cells did not affect IHERG, whereas the same intracellular concentration of astemizole caused a complete block. The results of the current study suggest that second-generation antihistamines display marked differences in their ability to block HERG K+ channels. Cetirizine in particular, which possesses more polar and smaller substituent groups attached to the tertiary amine compared with other antihistamines, lacks HERG-blocking properties, possibly explaining the absence of torsade de pointes ventricular arrhythmias associated with its therapeutical use.

The aim of the present study was to investigate whether K V 3.4 channel subunits are involved in neuronal death induced by neurotoxic ␤-amyloid peptides (A␤). In particular, to test this hypothesis, three main questions were addressed: 1) whether the A␤ peptide can up-regulate both the transcription/translation and activity of K V 3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in K V 3.4 expression and activity can be mediated by the nuclear factor-B (NF-B) family of transcriptional factors; and 3) whether the specific inhibition of K V 3.4 channel subunit reverts the A␤ peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that A␤ 1-42 treatment induced an increase in K V 3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the A␤ peptide caused an increase in I A current amplitude carried by K V 3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-B nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in K V 3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block A␤ 1-42 -induced increase in K V 3.4 K ϩ currents and to prevent cell death caused by A␤ 1-42 exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in K V 3.4 expression. As a whole, our data indicate that K V 3.4 channels could be a novel target for Alzheimer's disease pharmacological therapy.

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Background and Purpose-The sodium-calcium exchanger NCX1 represents a key mediator for maintaining [Na ϩ ] i and [Ca 2ϩ ] i in anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions. Methods-By means of Western blotting, patch-clamp electrophysiology, single-cell Fura-2 acetoxymethyl-ester microfluorometry, immunohistochemistry, and confocal microscopy, we investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion. The exchanger expression and activity were measured in primary microglia isolated ex vivo from the core region of adult rat brains 7 days after permanent middle cerebral artery occlusion and in cultured microglia under in vitro hypoxia. Results-One day after permanent middle cerebral artery occlusion, NCX1 protein expression was detected in some microglial cells adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after permanent middle cerebral artery occlusion, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells obtained from the core also displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca 2ϩ ] i increase induced by hypoxia was completely prevented. Key Words: cerebral ischemia Ⅲ IB4 Ⅲ microglia Ⅲ Na ϩ /Ca 2ϩ exchanger Ⅲ NCX1 Ⅲ pMCAO M icroglial cells are one of the first nonneuronal cells that respond to ischemic injury and in which, like occurs in most immune cells, the initiation of cellular responses, including morphological changes, proliferation, motility, phagocytosis, and increased synthesis of receptors and secretory products, involves modifications of calcium homeostasis. 1,2 In particular, long-lasting ionic alterations occurring in microglia in response to the ischemic injury play a crucial role in inducing and maintaining neurotrophic, inflammatory, and remodeling responses in the postischemic brain. 3 The sodium-calcium exchanger (NCX), an integral protein belonging to the superfamily of plasma membrane exchangers, represents a key mediator for maintaining intracellular [ 6 -8 NCX1 is broadly expressed in the heart, brain, and kidney, whereas NCX2 and NCX3 are exclusively expressed in the brain and skeletal muscle. 7,8 Among these 3 genes, the ubiquitous NCX1 is the most highly characterized member. 9 By contrast, in the brain, although isoform-specific cellular expression patterns suggest distinct functions for each of the 3 exchangers, 10 the physiological roles of the NCX1 gene product have not yet been as well defined as those in the heart. Conclusion-TheIn vitro studies provided e...

Changes in intracellular [Ca 2 þ ] i levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2 0 ,3 0 -cyclic nucleotide-3 0 -phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation. Oligodendrocytes, the myelin-forming cells of the CNS, arise from oligodendrocyte precursor cells (OPCs) that colonize both the gray and the white brain matter during development. 1 Although many OPCs differentiate into mature myelinating oligodendrocytes during the early and much later stages of human brain development, a considerable number of them do persist in the adult brain, and may provide a source of new oligodendrocytes, as well as protoplasmic astrocytes and neurons. 2,3 Because of their apparent stem-like characteristics, adult OPCs have recently gained much attention, for they are regarded as a potential reservoir of cells capable of self-renewal, differentiation, and re-myelination after CNS injury. 4 Thus, understanding how oligodendrocyte development proceeds and what factors govern the differentiation fate of OPCs is crucial to discover new effective therapeutic targets for de-myelinating diseases such as multiple sclerosis (MS).Changes in intracellular calcium levels have a critical role in OPC migration, lineage progression, and differentiation; furthermore, they are required for myelination and remyelination processes. [5][6][7] The Na þ /Ca 2 þ exchanger (NCX), 8 a transmembrane domain protein, which, by operating in a bidirectional way, couples the efflux of Ca 2 þ to the influx of Na þ into the cell or, vice versa, the influx of Ca 2 þ to the efflux of Na þ , is involved in the regulation of diverse neuronal and glial cell functions. 8,9 Furthermore, NCX is involved in regulating int...

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