Anoxia-induced NF-kappaB-dependent upregulation of NCX1 contributes to Ca2+ refilling into endoplasmic reticulum in cortical neurons.
OBJECTIVE
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.
Sirabella
Rossana
R
Fondazione IRCCS SDN, Naples, Italy.
Secondo
Agnese
A
Pannaccione
Anna
A
Scorziello
Antonella
A
Valsecchi
Valeria
V
Adornetto
Annagrazia
A
Bilo
Leonilda
L
Di Renzo
Gianfranco
G
Annunziato
Lucio
L
D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology*; D053454:Caspase 12 / Q000378:metabolism; D016923:Cell Death; D015687:Cell Hypoxia / Q000502:physiology*; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2; D005947:Glucose / Q000172:deficiency; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics*; D009474:Neurons / Q000378:metabolism*; D011247:Pregnancy; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis / Q000235:genetics*; D051996:Transcription Factor RelA / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation / Q000502:physiology
D005456:Fluorescent Dyes; D016328:NF-kappa B; D034741:RNA, Small Interfering; D019831:Sodium-Calcium Exchanger; D051996:Transcription Factor RelA; C110374:sodium-calcium exchanger 1; D053454:Caspase 12; D005947:Glucose; D016257:Fura-2