Activation of cPLA(2), PKC, and ERKs in the rat cerebral cortex during ischemia/reperfusion

Release of the excitotoxic amino acid, glutamate, into the extracellular sp ace during ischemia/reperfusion contributes to neuronal injury and death. T o gain insights into the signal transduction pathways involved in glutamate release we examined the time course of changes in enzyme levels and activi ties of cPLA(2), 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 cPLA(2) during 10-20 min of ischemia. Activity remained elevated at 10 min and 20 min of reperfusion, whereas cPLA levels had retur ned 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 PKC gamma levels were record ed in the particulate fraction during ischemia and reperfusion, and in the cytosolic fraction during ischemia. Western blot analysis with a phosphospe cific antibody for characterization of MAPK (ERKs) activation revealed sign ificantly increased phosphorylation of ERK1 and ERK2 in the particulate fra ction, of ERK2 in the cytosolic fraction, during ischemia and of both enzym es in the particulate and cytosolic fractions after 10 min of reperfusion. The relevance of the results to glutamate release is discussed.