Activation of cPLA2, PKC, and ERKs in the rat cerebral cortex during ischemia/reperfusion.
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.
Saluja
I
I
Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
O'Regan
M H
MH
Song
D
D
Phillis
J W
JW
D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002540:Cerebral Cortex / Q000201:enzymology* / Q000648:ultrastructure; D004789:Enzyme Activation; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D002546:Ischemic Attack, Transient / Q000201:enzymology*; D008297:Male; D010741:Phospholipases A / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015424:Reperfusion
D005230:Fatty Acids, Nonesterified; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A