Brain mu-calpain autolysis during global cerebral ischemia.
Proteolytic degradation of numerous calpain substrates, including cytoskeletal and regulatory proteins, has been observed during brain ischemia and reperfusion. In addition, calpain inhibitors have been shown to decrease degradation of these proteins and decrease postischemic neuronal death. Although these observations support the inference of a role for mu-calpain in the pathophysiology of ischemic neuronal injury, the evidence is indirect. A direct indicator of mu-calpain proteolytic activity is autolysis of its 80-kDa catalytic subunit, and therefore we examined the mu-calpain catalytic subunit for evidence of autolysis during cerebral ischemia. Rabbit brain homogenates obtained after 0, 5, 10, and 20 min of cardiac arrest were electrophoresed and immunoblotted with a monoclonal antibody specific to the mu-calpain catalytic subunit. In nonischemic brain homogenates the antibody identified an 80-kDa band, which migrated identically with purified mu-calpain, and faint 78- and 76-kDa bands, which represent autolyzed forms of the 80-kDa subunit. The average density of the 80-kDa band decreased by 25 +/- 4 (p = 0.008) and 28 +/- 9% (p = 0.004) after 10 and 20 min of cardiac arrest, respectively, whereas the average density of the 78-kDa band increased by 111 +/- 50% (p = 0.02) after 20 min of cardiac arrest. No significant change in the density of the 76-kDa band was detected. These results provide direct evidence for autolysis of brain mu-calpain during cerebral ischemia. Further work is needed to characterize the extent, duration, and localization of mu-calpain activity during brain ischemia and reperfusion as well as its role in the causal pathway of postischemic neuronal injury.
Neumar
R W
RW
Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Hagle
S M
SM
DeGracia
D J
DJ
Krause
G S
GS
White
B C
BC
D000818:Animals; D001329:Autolysis; D015153:Blotting, Western; D002545:Brain Ischemia / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D005260:Female; D007527:Isoenzymes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011817:Rabbits
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D002154:Calpain