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(Stroke. 2009;40:922.)
© 2009 American Heart Association, Inc.

Original Contributions

Anoxia-Induced NF-kB-Dependent Upregulation of NCX1 Contributes to Ca²⁺ Refilling Into Endoplasmic Reticulum in Cortical Neurons

Rossana Sirabella, PhD, MD; Agnese Secondo, PhD; Anna Pannaccione, PhD; Antonella Scorziello, PhD, MD; Valeria Valsecchi, PhD; Annagrazia Adornetto, PhD; Leonilda Bilo, MD; Gianfranco Di Renzo, MD Lucio Annunziato, MD

From the Division of Pharmacology (A. Secondo, A.P., A. Scorziello, V.V., A.A., L.B., G.D., L.A.), Department of Neuroscience, School of Medicine, "Federico II" University of Naples, Naples, Italy; and Fondazione IRCCS SDN (L.A., R.S.), Naples, Italy.

Correspondence to Lucio Annunziato, MD, Division of Pharmacology, Department of Neuroscience, "Federico II" University of Naples Via S. Pansini 5, 80131 Naples, Italy. E-mail lannunzi{at}unina.it

Background and Purpose— The 3 gene products of the Na⁺/Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ concentrations ([Ca²⁺]_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 Ca²⁺ 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 Ca²⁺ refilling in the endoplasmic reticulum, thus helping neurons to prevent endoplasmic reticulum stress during oxygen and glucose deprivation.

Key Words: [Ca²⁺]_i homeostasis • ER stress • Na⁺-Ca²⁺ exchanger • neuroprotection • OGD