The calpain proteolytic system in neonatal hypoxic-ischemia.
Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.
Blomgren
K
K
Institute of Anatomy and Cell Biology, University of Göteborg, Sweden. klas.blomgren@anatcell.gu.se
McRae
A
A
Elmered
A
A
Bona
E
E
Kawashima
S
S
Saido
T C
TC
Ono
T
T
Hagberg
H
H
D000818:Animals; D000831:Animals, Newborn; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002154:Calpain / Q000096:biosynthesis / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D005260:Female; D007839:Functional Laterality; D002534:Hypoxia, Brain / Q000378:metabolism*; D002546:Ischemic Attack, Transient / Q000378:metabolism*; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014158:Transcription, Genetic; D013049:Spectrin
D002135:Calcium-Binding Proteins; D002154:Calpain; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D013049:Spectrin; C033668:calpastatin