Postischemic reperfusion induces alpha-fodrin proteolysis by m-calpain in the synaptosome and nucleus in rat brain.
A membrane cytoskeletal protein, fodrin, is a substrate for a Ca2+-dependent protease, calpain. It remains unknown whether mu-calpain or m-calpain is involved in the proteolysis of either alpha- or beta-fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of mu-calpain or a significant change of m-calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca2+-dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE-cellulose column. Alpha- and beta-fodrins and m-calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of alpha-fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 microM, 1 ml, i.v.). The mu-calpain-specific fragment of beta-fodrin was not generated during ischemia-reperfusion, supporting the possibility of the involvement of m-calpain rather than mu-calpain in the alpha-fodrin proteolysis.
Fukuda
S
S
Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, Ube, Japan.
Harada
K
K
Kunimatsu
M
M
Sakabe
T
T
Yoshida
K
K
D000818:Animals; D001157:Arterial Occlusive Diseases / Q000150:complications; D002154:Calpain / Q000378:metabolism*; D002340:Carotid Artery Diseases / Q000150:complications; D002352:Carrier Proteins / Q000378:metabolism*; D002364:Caseins; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007022:Hypotension / Q000150:complications; D002546:Ischemic Attack, Transient / Q000201:enzymology / Q000209:etiology / Q000378:metabolism*; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D016548:Prosencephalon / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013574:Synaptosomes / Q000201:enzymology / Q000378:metabolism; D013049:Spectrin
D002154:Calpain; D002352:Carrier Proteins; D002364:Caseins; D008840:Microfilament Proteins; D013049:Spectrin