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Background and Purpose— Disturbance of endoplasmic reticulum (ER) Ca 2+ homeostasis causes neuronal cell injury in stroke. By contrast, ischemic preconditioning (IPC)—a brief sublethal ischemic episode affording tolerance to a subsequent ischemic insult—restores ER Ca 2+ homeostasis. Under physiological conditions, ER calcium content is continuously refilled by the interaction between the ER-located Ca 2+ sensor STIM (stromal interacting molecule) 1 and the plasma membrane channel ORAI1 (a structural component of the CRAC calcium channel)—2 key mediators of the store-operated calcium entry (SOCE) mechanism. However, the role played by ORAI1 and STIM1 in stroke and in IPC-induced neuroprotection during stroke remains unknown. Therefore, we explored whether ORAI1 and STIM1 might be involved in stroke pathogenesis and in IPC-induced neuroprotection. Methods— Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia and IPC were induced in rats by transient middle cerebral artery occlusion. Expression of ORAI1 and STIM1 transcripts and proteins and their immunosignals were detected by qRT-PCR, Western blot, and immunocytochemistry, respectively. SOCE and Ca 2+ release–activated Ca 2+ currents (I CRAC ) were measured by Fura-2 AM video imaging and patch-clamp electrophysiology in whole-cell configuration, respectively. Results— STIM1 and ORAI1 protein expression and immunosignals decreased in the ipsilesional temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion followed by reperfusion. Analogously, in primary hypoxic cortical neurons, STIM1 and ORAI1 transcript and protein levels decreased concurrently with SOCE and Ca 2+ release–activated Ca 2+ currents. By contrast, IPC induced SOCE and Ca 2+ release–activated Ca 2+ current upregulation, thereby preventing STIM1 and ORAI1 downregulation induced by oxygen and glucose deprivation+reoxygenation. Silencing of STIM1 or ORAI1 prevented IPC-induced tolerance and caused ER stress, as measured by GRP78 (78-kDa glucose regulated protein) and caspase-3 upregulation. Conclusions— ORAI1 and STIM1, which participate in SOCE, take part in stroke pathophysiology and play an important role in IPC-induced neuroprotection.

Background and Purpose— The 3 gene products of the Na + /Ca 2+ exchanger (NCX), viz, NCX1, NCX2, and NCX3, may play a pivotal role in the pathophysiology of brain ischemia. The aim of this study was to investigate the transductional and posttranslational mechanisms involved in the expression of these isoforms during oxygen and glucose deprivation and their role in endoplasmic reticulum Ca 2+ refilling in cortical neurons. Methods— NCX1, NCX2, and NCX3 transcript and protein expression was evaluated in primary cortical neurons by reverse transcriptase–polymerase chain reaction and Western blot. NCX currents (I NCX ) and cytosolic Ca 2+ concentrations ([Ca 2+ ] i ) were monitored by means of patch-clamp in whole-cell configuration and Fura-2AM single-cell video imaging, respectively. Results— Exposure of cortical neurons to 3 hours of oxygen and glucose deprivation yielded dissimilar effects on the 3 isoforms. First, it induced an upregulation in NCX1 transcript and protein expression. This change was exerted at the transcriptional level because the inhibition of nuclear factor kappa B translocation by small interfering RNA against p65 and SN-50 prevented oxygen and glucose deprivation-induced NCX1 upregulation. Second, it elicited a downregulation of NCX3 protein expression. This change, unlike NCX1, was exerted at the posttranscriptional level because it was prevented by the proteasome inhibitor MG-132. Finally, we found that it significantly increased I NCX both in the forward and reverse modes of operation and promoted an increase in ER Ca 2+ accumulation. Interestingly, such accumulation was prevented by the silencing of NCX1 and the NCX inhibitor CB-DMB that triggered caspase-12 activation. Conclusions— These results suggest that nuclear factor kappa B-dependent NCX1 upregulation may play a fundamental role in Ca 2+ refilling in the endoplasmic reticulum, thus helping neurons to prevent endoplasmic reticulum stress during oxygen and glucose deprivation.

Background and Purpose— The sodium–calcium exchanger NCX1 represents a key mediator for maintaining [Na + ] i and [Ca 2+ ] 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 2+ ] i increase induced by hypoxia was completely prevented. Conclusion– The upregulation of NCX1 expression and activity observed in microglia after permanent middle cerebral artery occlusion suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.