Diphosphorylation and involvement of extracellular signal-regulated kinases (ERK1/2) in glutamate-induced apoptotic-like death in cultured rat cortical neurons.
Glutamate-induced excitotoxicity, with certain characteristics of apoptosis, has been implicated in a variety of neuronal degenerative disorders. In some physiological cases, extracellular signal-regulated kinases (ERK1/2) are activated by stimulation of glutamate receptors. In the present study, the activation (diphosphorylation) and role of ERK1/2 in glutamate-induced apoptotic-like death in cultured cortical neurons were investigated. Protein levels and activation (diphosphorylation) levels of ERK1/2 were examined by Western immunoblot, probed with anti-ERK1/2 and anti-active (diphosphorylated) ERK1/2 antibodies, respectively. Apoptotic-like death was determined by DAPI staining. Before a remarkable increase of apoptotic-like cell death was observed at 9-18 h after 15 min exposure to 50 microM glutamate, diphosphorylation levels of ERK1/2 were rapidly increased, peaked at 5-15 min of the exposure, and reverted to sham control level 3 h after the exposure, while the protein levels of ERK1/2 were unaffected. The glutamate concentration effective for inducing apoptotic-like cell death was correlated with that for inducing ERK1/2 diphosphorylation. Both ERK1/2 diphosphorylation and the apoptotic-like cell death were largely prevented by MK-801, a specific NMDA receptor (a subtype receptor of glutamate) antagonist, or the elimination of extracellular Ca(2+) with EGTA. PD98059, a specific inhibitor of ERK1/2 kinase, completely inhibited ERK1/2 diphosphorylation and partially inhibited the apoptotic-like cell death. These results suggest that largely via NMDA receptor-mediated influx of extracellular Ca(2+), ERK1/2 were rapidly and transiently activated and were involved in glutamate-induced apoptotic-like death in cultured rat cortical neurons.
Jiang
Q
Q
Research Center of Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, China.
Gu
Z
Z
Zhang
G
G
Jing
G
G
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D018929:Cell Culture Techniques; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects* / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D005419:Flavonoids; D009498:Neurotoxins; D018698:Glutamic Acid; D004533:Egtazic Acid; D016291:Dizocilpine Maleate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one