Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0022116 (ischemia)
 91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calpain activity was measured in the various subfractions of rat myocardia after global ischemia for 60 min or after ischemia followed by 30 min of reperfusion after the chromatographic separation of mu- and m-calpains. The activity of m-calpain after ischemia and that of mu-calpain after reperfusion were both higher than that in the control. The activity of the endogenous calpain inhibitor calpastatin in 10,000 x g supernatant was decreased after both ischemia and ischemia-reperfusion. The increase in m- and mu-calpain activities was suppressed by pre-ischemic perfusion with a synthetic calpain inhibitor, transepoxysuccinyl-L-leucylamido (4-guanidino) butane (E64d, 100 micrograms/ml). After reperfusion, the calpain activity in the 10,000 x g pellet was also increased, which was inhibited by pre-ischemic perfusion with E64d or dimethylsulfoxide (a solvent for E64d) or by reperfusion with 1 mmol/L ethyleneglycol bis (beta-aminoethylether)-N, N, N', N'-tetraacetic acid. SDS-polyacrylamide gel electrophoresis revealed the proteolysis of several proteins, including fodrin, in the 10,000 x g and 100,000 x g pellet fractions after ischemia and reperfusion, some of which were confirmed to be in vitro substrates of calpain. The creatine kinase release during the reperfusion was also partially inhibited by E64d or dimethylsulfoxide. Thus, calpain activity in the soluble or particulate fractions was altered during ischemia or reperfusion, and appeared to be implicated in the proteolysis of the membrane proteins, which may contribute to myocardial injury.
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PMID:Calpain is implicated in rat myocardial injury after ischemia or reperfusion. 775 44

Inactivation of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been implicated in ischemia-reperfusion injury to heart and other organs. Because oxidation of many proteins makes them more susceptible to degradation by intracellular proteinases, we studied the effects of several such proteinases on native and H2O2-oxidized preparations of Na+/K(+)-ATPase from canine kidney (containing alpha 1 isoform of the catalytic subunit) and rat axolemma (containing alpha 2 and alpha 3 isoforms). Lysosomal cathepsin D degraded the native and the oxidized preparations at acid pH, but it was significantly more effective against the oxidized forms. m-Calpain had little or no effect on the native Na+/K(+)-ATPase preparations, but it digested the oxidized alpha-subunits of the axolemma and the kidney enzymes. mu-Calpain's effects were similar to those of m-calpain. Multi-catalytic proteinase which is known to degrade a large number of oxidized proteins, did not affect the native or the oxidized forms of Na+/K(+)-ATPase. The findings suggest that (a) during oxidative stress there may be accelerated degradation of the oxidatively damaged Na+/K(+)-ATPase, either through internalization and transport to lysosomes, or by the action of calpains at the membrane; and (b) those isoforms of the enzyme that are more sensitive to oxidants are more susceptible to degradation by the above processes.
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PMID:Different sensitivities of native and oxidized forms of Na+/K(+)-ATPase to intracellular proteinases. 820 42

To examine whether calpain is activated during ischemic or reperfusion injury, we measured calpain activity of the subfractions of rat myocardia after global ischemia for 60 min or the ischemia followed by 30 min reperfusion by the Langendorff procedure. The myocardial homogenate was fractionated into 600 x g, 10,000 x g and 100,000 x g pellet fractions as well as 10,000 x g supernatant fraction. The supernatant fraction was further subjected to DEAE cellulose and phenyl-Sepharose chromatographies to separate mu- and m-calpains. The m-calpain activity of the DEAE fractions after global ischemia for 60 min was higher but that after ischemia-reperfusion was lower than that of the control. On the other hand, the ischemia-reperfusion but not ischemia by itself raised the calpain activity of the phenyl-Sepharose fraction (mu-calpain) and the 10,000 x g pellet measured at 100 microM and 5 mM Ca2+. Treatment with verapamil but not with ryanodine during ischemia attenuated the increase in m-calpain activity. A dot-blotting analysis of calpain antigenicity showed a decrease in soluble but no change in the particulate fractions after ischemia-reperfusion. An immunoblotting technique did not detect proteolysis of the calpain 80-kDa subunit. These observations suggest that calpain is activated by Ca2+ influx during ischemia and reperfusion without gross changes in its amount. Some unknown processes other than translocation or autolysis are thought to be involved in the alterations.
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PMID:Calpain activity alters in rat myocardial subfractions after ischemia or reperfusion. 835 52

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.
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PMID:The calpain proteolytic system in neonatal hypoxic-ischemia. 936 79

The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20 min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20 min followed by reperfusion for 30 min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. Mu-calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of mu- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.
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PMID:Downregulation of calpastatin in rat heart after brief ischemia and reperfusion. 939 77

We have determined the effects of the calpain inhibitors AK275 and AK295 upon purified m-calpain and calcium-mediated degradation of neurofilament protein (NFP) in rat spinal cord in vitro. After incubation, the soluble radioactivity and/or extent of myelin basic protein (MBP) or NFP degradation was determined. Fifty percent of caseinolytic activity was inhibited by both inhibitors at 0.6 microM concentration, while more than 90% inhibition was seen at 1.6 microM. In contrast, 37% and 64% inhibition of MBP degradation was seen with AK295 and AK275, respectively, at 10 microM concentration. The extent of NFP degradation in spinal cord was quantified from immunoblot enhanced chemiluminescence. The calcium-mediated breakdown of NFP was inhibited by both AK275 and AK295, and the inhibition was dose-dependent. A 50% inhibition of NFP degradation was seen with AK295 at 10 microM and was almost completely inhibited at 25-50 microM. AK295 was slightly more potent than AK275. These studies suggest that these potent calpain inhibitors may be used therapeutically to provide neuroprotection in vivo in experimental central nervous system trauma and ischemia.
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PMID:New inhibitors of calpain prevent degradation of cytoskeletal and myelin proteins in spinal cord in vitro. 946 75

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.
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PMID:Postischemic reperfusion induces alpha-fodrin proteolysis by m-calpain in the synaptosome and nucleus in rat brain. 960 18

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.
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PMID:Dual response of calpain to rat brain postdecapitative ischemia. 964 72

Overactivated calpain might be a key factor in destruction of cytoskeletal proteins involved in the pathophysiology of ischemia and disorders like Alzheimer's disease. Therapeutic effects imply the possible interference of Cerebrolysin (Ebewe Arzneimittel, Austria) with these molecular events. In this work several in vitro methods have been applied to investigate the interaction between Cerebrolysin and calpain [Enzyme Commission (EC) number: 3.4.22.17]. A conventional caseinolytic assay beside two flourimetric assays using a synthetic peptide substrate and a fluorescence labelled cytoskeletal protein [microtubule-associated protein 2 labelled with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (MAP2-DTAF)] respectively for a highly sensitive fluorimetric calpain activity assay were applied for kinetic analysis. The caseinolytic assay showed that the drug inhibits both mu- and m-calpain and to a significantly lower extent also trypsin [Enzyme Commission (EC) number: 3.4.21.1] and papain [Enzyme commission (EC) number: 3.4.22.6]. Dialysis experiments revealed Cerebrolysin mediated calpain inhibition to be reversible. Kinetic analysis exhibited a non-competitive, or tight-binding competitive, mode of inhibition. This latter mode, substantiated by serial dilution experiments, and the likely existence of calpastatin in a brain derivative suggests the occurrence of calpastatin fragments or calpastatin-like fragments in Cerebrolysin. The clearly competitive inhibition of trypsin by the drug indicates distinct mechanisms and active components against different proteases.
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PMID:Inhibitory effect of a brain derived peptide preparation on the Ca++-dependent protease, calpain. 1084 56

Neuronal cytoskeletal proteins like the microtubule associated protein 2 (MAP2) are objected to pathological proteolysis in case of Alzheimer's disease and brain ischemia. The neurotrophic peptidergic drug Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is produced by a standardized enzymatic break-down of lipid free porcine brain proteins. Cerebolysin protected MAP2 in primary neuronal cultures after a brief histotoxic hypoxia and in a rat model of acute brain ischemia. Furthermore the drug was shown to inhibit the proteases mu- and m-calpain dose dependently in several cell free protease activity assays. The question if the higher MAP2 levels are due to an alleviation of proteolysis, to a higher synthesis rate or both is addressed in the current investigation: Monitoring the MAP2 content of primary neuronal cell cultures over a period of eight days revealed MAP2 to reach a peak level on day six in vitro followed by a degradation phase. In other experiments the protein synthesis of Cerebrolysin treated and untreated cells was blocked with cycloheximide at that moment when all cells exhibited the same MAP2 content. After the following MAP2 degradation phase--i.e. after eight days in vitro--the MAP2 contents were determined by western blotting. Cerebrolysin treated cells contained more MAP2 than untreated controls proving that the drug protects MAP2 independently from de novo synthesis, although further work is in progress to investigate if the drug supplementary boosts this effect by increasing MAP2 synthesis.
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PMID:A brain derived peptide preparation reduces the translation dependent loss of a cytoskeletal protein in primary cultured chicken neurons. 1096 38


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