calpastatin and Ischemic-Attack--Transient

The activation of the [Ca(2+)]-dependent cysteine protease calpain plays an important role in ischemic injury. Here, the levels of two calpain-specific substrates, p35 protein and eukaryotic initiation factor 4G (eIF4G), as well as its physiological regulator calpastatin, were investigated in a rat model of transient global cerebral ischemia with or without ischemic tolerance (IT). Extracts of the cerebral cortex, whole hippocampus and hippocampal subregions after 30 min of ischemia and different reperfusion times (30 min and 4 h) were used. In rats without IT, the p35 levels slightly decreased after ischemia or reperfusion, whereas the levels of p25 (the truncated form of p35) were much higher than those in sham control rats after ischemia and remained elevated during reperfusion. The eIF4G levels deeply diminished after reperfusion and the decrease was significantly greater in CA1 and the rest of the hippocampus than in the cortex. By contrast, the calpastatin levels did not significantly decrease during ischemia or early reperfusion, but were upregulated after 4 h of reperfusion in the cortex. Although IT did not promote significant changes in p35 and p25 levels, it induced a slight increase in calpastatin and eIF4G levels in the hippocampal subregions after 4 h of reperfusion.

Topics: Animals; Brain; Brain Chemistry; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Eukaryotic Initiation Factor-4G; Hippocampus; HSP70 Heat-Shock Proteins; Ischemic Attack, Transient; Ischemic Preconditioning; Mice; Phosphotransferases; Rats; Rats, Wistar; Reperfusion Injury; Tissue Extracts

Effects of ischemia on the content of a ulinastatin (UT)-like substance in the murine cerebral cortex and hippocampus were studied. At 24 h post-ischemia, a significant (p < 0.05) decrease in the content of UT-like substance in the hippocampus but not the cerebral cortex and a concurrent increase in the activity of micro-calpain were observed. In in vitro experiments, a decrease was registered in the content of UT-like substance in the hippocampus in the presence of calcium. This decrease was inhibited by both EDTA and calpastatin treatments. These results implicate the destruction of UT-like substance by micro-calpain in the ischemic hippocampus.

Topics: Animals; Calcium Chloride; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Edetic Acid; Glycoproteins; Hippocampus; Ischemic Attack, Transient; Kinetics; Male; Mice; Mice, Inbred ICR; Trypsin Inhibitors

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37 degrees C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 micromol/L calpastatin or 10 micromol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% +/- 1.1% to 15.8% +/- 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Eukaryotic Initiation Factor-4G; Female; Ischemic Attack, Transient; Kinetics; Male; Peptide Initiation Factors; Rabbits; Rats; Reperfusion; Subcellular Fractions

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.

Topics: Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytosol; DNA Primers; Female; Functional Laterality; Hypoxia, Brain; Ischemic Attack, Transient; Male; Microfilament Proteins; Nerve Tissue Proteins; Rats; Rats, Wistar; Transcription, Genetic

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery plus 7.7% O2 for 2 h) and allowed to recover for 0 min, 30 min or 20 h. The calpain and calpastatin activities were assayed in subcellular fractions of the ipsilateral, hypoxic-ischemic and the contralateral, hypoxic hemisphere. An upregulation of calpain activity occurred in the hypoxic hemisphere, both in the major, cytosolic fraction and in the hypotonic, membrane associated fraction (110% and 133% of controls, respectively). The hypoxic-ischemic hemisphere displayed a decrease in calpain activity in the cytosolic fraction but an increase in the hypotonic fraction (90% and 111% of controls, respectively). The changes in calpastatin activity were less pronounced. This indicates that an upregulation of calpain activity occurs in parallel with development of hypoxic-ischemic damage. However, this upregulation is not necessarily coupled to development of injury as lesions are not seen in the hypoxic hemisphere.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Humans; Hypoxia, Brain; Infant, Newborn; Ischemic Attack, Transient; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Up-Regulation

Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, while contraction was induced in the normal canine basilar artery by a local application of KCl or serotonin after transclival exposure. The control animals were injected with saline instead of fresh blood. The activation of mu-calpain, a Ca(++)-dependent neutral protease, in the basilar artery was studied by evaluating the conversion from its inactivated into its activated form on immunoblots. In addition, the activity of calpastatin, an intrinsic inhibitor of calpain, in the basilar artery was determined by assay. The majority of the mu-calpain was inactivated in the control group. In the spastic group, mu-calpain was generally activated markedly in the early stage of vasospasm and moderately thereafter. The contraction induced by KCl or serotonin application was classified into the early phasic and the later tonic stages; mu-calpain was usually activated in the phasic stage and inactivated in the tonic stage. Calpastatin activity was significantly decreased during vasospasm, whereas it was not significantly changed in KCl- or serotonin-induced contraction. The final activity of mu-calpain results from the balance of mu-calpain and calpastatin. This suggests that mu-calpain activity was enhanced continuously in the spastic group and transiently in the KCl or serotonin group, and that the continuous activation of mu-calpain during vasospasm probably induced more proteolytic changes compared to those in the KCl or serotonin group.

Topics: Animals; Basilar Artery; Calcium-Binding Proteins; Calpain; Dogs; Immunoblotting; In Vitro Techniques; Ischemic Attack, Transient; Potassium Chloride; Serotonin