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 s ome physiological cases, extracellular signal-regulated kinases (ERK1/2) ar e activated by stimulation of glutamate receptors. In the present study, th e activation (diphosphorylation) and role of ERK1/2 in glutamate-induced ap optotic-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 (diphosphoryla ted) ERK1/2 antibodies, respectively. Apoptotic-like death was determined b y DAPI staining. Before a remarkable increase of apoptotic-like cell death was observed at 9-18 h after 15 min exposure to 50 mu M glutamate, diphosph orylation levels of ERK1/2 were rapidly increased, peaked at 5-15 min of th e exposure, and reverted to sham control level 3 h after the exposure, whil e 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 th e apoptotic-like cell death were largely prevented by MK-801, a specific NM DA receptor (a subtype receptor of glutamate) antagonist, or the eliminatio n of extracellular Ca2+ with EGTA. PD98059, a specific inhibitor of ERK1/2 kinase, completely inhibited ERK1/2 diphosphorylation and partially inhibit ed the apoptotic-like cell death. These results suggest that largely via NM DA receptor-mediated influx of extracellular Ca2+, ERK1/2 were rapidly and transiently activated and were involved in glutamate-induced apoptotic-like death in cultured rat cortical neurons. (C) 2000 Elsevier Science B.V. All rights reserved.