Release of the excitotoxic
amino acid,
glutamate, into the extracellular space during
ischemia/reperfusion contributes to neuronal injury and death. To gain insights into the signal transduction pathways involved in
glutamate release we examined the time course of changes in
enzyme levels and activities of cPLA2, PKC and ERKs in the rat cerebral cortex after four vessel (4VO)
ischemia followed by reperfusion. Measurement both by enzymatic assay and Western blot analysis showed significant increases in the activity and
protein levels of cPLA2 during 10-20 min of
ischemia. Activity remained elevated
at 10 min and 20 min of reperfusion, whereas cPLA levels had returned to base line levels after 20 min of reperfusion. PKC activity increased significantly in the particulate, but not in the cytosolic, fractions both during
ischemia and reperfusion. Increases in PKCgamma levels were recorded in the particulate fraction during
ischemia and reperfusion, and in the cytosolic fraction during
ischemia. Western blot analysis with a
phosphospecific antibody for characterization of MAPK (ERKs) activation revealed significantly increased phosphorylation of ERK1 and ERK2 in the particulate fraction, of ERK2 in the cytosolic fraction, during
ischemia and of both
enzymes in the particulate and cytosolic fractions after 10 min of reperfusion. The relevance of the results to
glutamate release is discussed.