Depolarization induces NR2A tyrosine phosphorylation and neuronal apoptosis.
BACKGROUND
Cytosol Ca2+ overload plays a vital role in ischemic neuronal damage, which is largely contributed by the Ca2+ influx through L-type voltage-gated calcium channels (L-VGCCs) and N-methyl-D-aspartate (NMDA) type glutamate receptors. In this article, L-VGCCs were activated by depolarization to investigate the cross-talk between NMDA receptors and L-VGCCs.


METHODS
Depolarization was induced by 20 minutes incubation of 75 mM KCl in cultured rat cortical neuron. Apoptosis-like neuronal death was detected by DAPI staining. Tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A), interactions of Src and NR2A were detected by immunoblot and immunoprecipitation.


RESULTS
Depolarization induced cortical neuron apoptosis-like cell death after 24 hours of restoration. The apoptosis was partially inhibited by 5 mM EGTA, 100 μM Cd2+, 10 μM nimodipine, 100 μM genistein, 20 μM MK-801, 2 μM PP2 and combined treatment of nimodipine and MK-801. NR2A tyrosine phosphorylation increased after depolarization, and the increase was inhibited by the drugs listed above. Moreover, non-receptor tyrosine kinase Src bound with NR2A after depolarization and restoration. The binding was also inhibited by the drugs listed above.


CONCLUSIONS
The results indicated that depolarization-induced neuronal death might be due to extracellular Ca2+ influx through L-VGCCs and subsequently Src activationmediated NR2A tyrosine phosphorylation.
Research Center for Biochemistry & Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, 221002, China.
Yong
Y
Liu
Jing-Zhi
JZ
Yan
Ying-Hui
YH
Gu
Wei
W
Wang
Yan-Yan
YY
Zong
Xiao-Yu
XY
Hou
Guang-Yi
GY
Zhang
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D047468:Immunoprecipitation; D007211:Indoles; D007529:Isoflavones / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009553:Nimodipine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
C412373:AG 1879; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D007211:Indoles; D007529:Isoflavones; C120997:NR2A NMDA receptor; D011743:Pyrimidines; D016194:Receptors, N-Methyl-D-Aspartate; C040641:genistin; D014443:Tyrosine; C007293:DAPI; D009553:Nimodipine; D011189:Potassium Chloride; D016291:Dizocilpine Maleate; D019061:src-Family Kinases