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PubMed1997

Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

Antibody Specificity↗
PubMed1995

Casein zymography: a method to study mu-calpain, m-calpain, and their inhibitory agents.

A zymographic assay for calpains in nondenaturing casein-containing polyacrylamide gels was developed. Calpain samples were run into the polyacrylamide gels by electrophoresis using a Tris-glycine buffer containing 1 mM EGTA to stabilize calpains. Upon completion of the electrophoresis, the gels were washed and incubated in a calpain activation buffer containing 1-4 mM calcium and 10 mM dithiothreitol for 20-24 h. After staining of the casein gels with Coomassie blue G250, both mu-calpain and m-calpain showed up as clearing bands. The amount of calpain loaded was proportional to the brightness of the clearing band. m-calpain can be easily distinguished from mu-calpain due to its higher mobility in the gel. Irreversible inhibitor (e.g., E64c) or tight-binding calmidazolium-treated mu-calpain remained inactive in the casein zymogram, whereas reversible inhibitor (e.g., calpain inhibitor I) was released from the protease by migration and dilution, lifting its inhibition. Crude homogenate of cultured cells (erythrocytes, Molt-4 and cerebrocortical neurons) or tissue (rat brain) can be directly analyzed for the presence of calpain isoforms despite the presence of endogenous calpastatin. Using this technique, mu-calpain activity in Molt-4 cells was found to decrease progressively with A23187 treatment, as a reflection of autolytic inactivation.

Animals↗
PubMed1992

A comparison of the intracellular distribution of mu-calpain, m-calpain, and calpastatin in proliferating human A431 cells.

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.

Antibodies, Monoclonal↗
PubMed1993

Cloning the partial cDNAs of mu-calpain and m-calpain from porcine skeletal muscle.

Calpains are non-lysosomal proteases involved in myofibrillar protein degradation. To facilitate studying the expression of the porcine calpain genes and their influence on protein accretion, we have cloned partial cDNAs for mu- and m-calpain from porcine skeletal muscle via PCR amplification. A 289 bp fragment for mu-calpain and a 629 bp fragment for m-calpain were cloned into the EcoRV site of pBluescript II KS+ vector. The nucleotide sequence for porcine mu-calpain and m-calpain were 92% and 90% identical to corresponding regions of rabbit mu- and m-calpain, respectively. The deduced amino acid sequences for both mu- and m-calpain share 94% identity with respective rabbit mu- and m-calpains. Isoform specificity was validated by Southern hybridization of mu- and m-calpain probes with cloned mu- and m-calpain fragments and Northern hybridization with pig muscle mRNA. These clones will be used to evaluate the role of calpain expression in muscle hypertrophy.

Amino Acid Sequence↗
PubMed2003

Calpain is activated in experimental uremia: is calpain a mediator of uremia-induced myocardial injury?

BACKGROUND: The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia. METHODS: Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time. RESULTS: In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01). CONCLUSIONS: In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.

Acrylates↗
PubMed1998

Subcellular localization and duration of mu-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study.

Casein zymographic assays were performed to identify changes in mu-calpain and m-calpain activity in naive, sham-injured, and injured rat cortex at 15 minutes, 3 hours, 6 hours, and 24 hours after unilateral cortical impact brain injury. Cortical samples ipsilateral and contralateral to the site of injury were separated into cytosolic and total membrane fractions. Marked increases in mu-calpain activity in cytosolic fractions in the ipsilateral cortex occurred as early as 15 minutes, became maximal at 6 hours, and decreased at 24 hours to levels observed at 15 minutes after injury. A similar temporal profile of cytosolic mu-calpain activity in the contralateral cortex was observed, although the increases in the contralateral cortex were substantially lower than those in the ipsilateral cortex. Differences were also noted between cytosolic and total membrane fractions. The detection of a shift in mu-calpain activity to the total membrane fraction first occurred at 3 hours after traumatic brain injury and became maximal at 24 hours after traumatic brain injury. This shift in mu-calpain activity between the two fractions could be due to the redistribution of mu-calpain from the cytosol to the membrane. m-Calpain activity was detected only in cytosolic fractions. m-Calpain activity in cytosolic fractions did not differ significantly between ipsilateral and contralateral cortices, and increased in both cortices from 15 minutes to 6 hours after injury. Relative magnitudes of m-calpain versus mu-calpain activity in cytosolic fractions differed at different time points after injury. These studies suggest that traumatic brain injury can activate both calpain isoforms and that calpain activity is not restricted to sites of focal contusion and cell death at the site of impact injury but may represent a more global response to injury.

Animals↗
PubMed2002

Calpain inhibition by cerebrospinal fluid and effects of calpain on intrathecal nerve tissue.

STUDY DESIGN: The effects of calpain on intrathecal nerve tissue in the rabbit were investigated. OBJECTIVE: To evaluate the chemonucleolytic side effect of calpain on nerve tissue in the event of accidental intrathecal calpain injection. SUMMARY OF BACKGROUND DATA: Calpain has a degradative effect on proteoglycans, and as previously shown, it is associated with chemonucleolytic action in the rabbit. However, its effect on nerve tissue in the event of accidental intrathecal injection is not clear. METHODS: The inhibitory activity of cerebrospinal fluid against calpain was measured in human cerebrospinal fluid using mu-calpain, and in different cerebrospinal fluid fractions separated by molecular filtration. The presence of the endogenous calpain inhibitor, calpastatin, in human cerebrospinal fluid was examined by Western blotting with anticalpastatin antibody. After intrathecal application of calpain in rabbits, the spinal cord nerve tissue was examined by light microscopy. RESULTS: Cerebrospinal fluid inhibited the enzyme reaction of calpain at its normal concentration. Immunoblotting with anticalpastatin antibody did not yield positive staining. After the intrathecal application of calpain, there was no evidence of degeneration in the nerve tissue of the spinal cord. CONCLUSIONS: This study suggests that in the event of accidental intrathecal injection of calpain for chemonucleolysis, the enzyme activity of calpain will be neutralized by cerebrospinal fluid, and the calpain should not cause unwanted side effects in chemonucleolysis.

Adolescent↗
PubMed2006

Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction.

Calpains, Ca(2+)-activated cysteine proteases, are cytosolic enzymes implicated in numerous cellular functions and pathologies. We identified a mitochondrial Ca(2+)-inducible protease that hydrolyzed a calpain substrate (SLLVY-AMC) and was inhibited by active site-directed calpain inhibitors as calpain 10, an atypical calpain lacking domain IV. Immunoblot analysis and activity assays revealed calpain 10 in the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix fractions. Mitochondrial staining was observed when COOH-terminal green fluorescent protein-tagged calpain 10 was overexpressed in NIH-3T3 cells and the mitochondrial targeting sequence was localized to the NH(2)-terminal 15 amino acids. Overexpression of mitochondrial calpain 10 resulted in mitochondrial swelling and autophagy that was blocked by the mitochondrial permeability transition (MPT) inhibitor cyclosporine A. With the use of isolated mitochondria, Ca(2+)-induced MPT was partially decreased by calpain inhibitors. More importantly, Ca(2+)-induced inhibition of Complex I of the electron transport chain was blocked by calpain inhibitors and two Complex I proteins were identified as targets of mitochondrial calpain 10, NDUFV2, and ND6. In conclusion, calpain 10 is the first reported mitochondrially targeted calpain and is a mediator of mitochondrial dysfunction through the cleavage of Complex I subunits and activation of MPT.

3T3 Cells↗
PubMed2006

Calpain-specific proteolysis in primate retina: Contribution of calpains in cell death.

PURPOSE: One of the leading causes of blindness is retinal damage caused by the high intraocular pressure (IOP) in glaucoma. Previous studies in rats have suggested that the proteolytic enzyme calpain (EC 3.4.22.17) is involved in retinal cell death during ischemia and in acute high IOP. Ubiquitous, calcium-activated calpain-1 and -2 from monkey retina are highly homologous to rat calpains, although expression patterns in variants of tissue-specific calpain-3 are different between monkey and rodent retinas. Thus, the purpose of the present study was to investigate the involvement of calpain-induced proteolysis in retinal cell death in primates. METHODS: Calpain involvement in a simulated pathologic condition was examined by incubating monkey retinas in hypoxic conditions (95% N2 and 5% CO2) in RPMI medium without glucose. Endogenous tissue calpains were also directly activated in monkey and human retinal soluble proteins by incubating with 2.5 mM calcium. The resultant proteolysis of monkey retinal proteins was assessed by 2D electrophoresis (2-DE). RESULTS: In hypoxic retina, leakage of lactate dehydrogenase (LDH) from retinas into the medium increased, indicating cell death. LDH leakage was partially inhibited by the calpain inhibitor SJA6017. Calpain autolysis was observed, and the calpain-preferred substrate alpha-spectrin was proteolyzed. In retinal soluble proteins incubated with calcium, a total of 15 spots from 2-DE of retinal soluble proteins were identified by mass spectrometry. Proteolysis of major proteins, vimentin, beta-tubulin, alpha-enolase, and Hsp70 were confirmed by immunoblot analysis. Activation of calpains and proteolysis of these substrates were inhibited by the calpain-specific inhibitor SJA6017. CONCLUSIONS: Taken together, these results suggested that calpain activation in primate retinas could play an important role in cell death during hypoxia caused by elevated IOP from glaucoma.

Adult↗
PubMed1999

Quantification of ovine and bovine calpain I, calpain II, and calpastatin mRNA by ribonuclease protection assay.

We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.

Animals↗
PubMed1983

Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney.

Two molecular species of calpain (Ca2+-dependent cysteine proteinases) were concurrently purified from rat kidney, both to homogeneity. Calpain I and calpain II having low and high Ca2+ requirements, respectively, were clearly separated on DEAE-cellulose chromatography at pH 7.5, and thereafter they were purified by separate but almost identical procedures which included (NH4)2SO4 fractionation and successive chromatographies on TSK-Gel G 3000 SWG, blue Sepharose CL-6B, and DEAE-Bio-Gel A. The purification folds and activity yields were 6170-fold and 17.8% for calpain I and 4160-fold and 11.9% for calpain II. Ca2+ concentrations for half-maximal activation were 2 microM for calpain I and 200 microM for calpain II. The specific activity of calpain II on casein as the substrate was more than twice higher than that of calpain I. Both enzymes are heterodimers, each composed of 80,000-Da and 25,000-Da subunits. The amino acid compositions of calpain I and calpain II are very similar but not identical. Calpain II is more acidic (pI 4.6) than calpain I (pI 5.3). This paper is the first to describe parallel isolation and characterization of low and high Ca2+-requiring proteases from one single nonmuscular tissue.

Amino Acids↗
PubMed1993

Calpastatin has two distinct sites for interaction with calpain--effect of calpastatin fragments on the binding of calpain to membranes.

Calpain, a ubiquitously distributed intracellular cysteine protease in animals, is thought to be activated at the cell membrane in the presence of micromolar levels of calcium ions. Calpastatin, the sole specific proteinaceous inhibitor for calpain, inhibits not only proteolytic activity, but also the binding of calpain to cell membranes. The inhibition of calpain binding to membranes by calpastatin fragments was analyzed. A calpastatin peptide containing the inhibitory sequence did not inhibit the binding of calpain to membranes. On the other hand, binding was prevented by a fragment without the activity to inhibit proteolytic activity. These data indicate that calpain binds to cell membranes through a site (regulatory site) other than the active site and that calpastatin inhibits the binding of calpain to cell membranes via a site (regulatory inhibition site) other than the inhibitory sequence. Calpain and calpastatin can undergo independent interactions: interaction between the catalytic site of calpain and the inhibitory sequence of calpastatin, and interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin. The interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin is essential for the regulation of calpain activity.

Amino Acid Sequence↗
PubMed1997

Immunological analysis of two calpain-like Ca2+-dependent proteinases from lobster striated muscles: relationship to mammalian and Drosophila calpains.

Lobster skeletal muscles contain four Ca2+-dependent cysteine proteinases (CDPs I, IIa, IIb, and III) that degrade myofibrillar proteins. Lobster CDPs share many properties with calpains from vertebrate tissues, but differ in native mass and subunit composition. Recently, cDNAs encoding a calpain-like protein (Dm-calpain; 91.5 or 94 kDa) have been isolated from fruit fly, Drosophila melanogaster. To further clarify the relationship between invertebrate CDPs and mammalian calpains, antibodies specific for mu-, m-, p94 (nCL-1), and Dm-calpains and lobster CDP IIb (native M(r) 195,000, subunit M(r) 95,000) were used in immunoblots to test for antigenic cross-reactivity. No common epitopes were found between CDP IIb and vertebrate calpains. However, polyclonal antibodies to CDP IIb cross-reacted strongly with a C-terminal 70-kDa portion of Dm-calpain expressed in Escherichia coli. Conversely, polyclonal antibodies to Dm-calpain recognized CDP IIb. A second CDP, CDP IIa (native M(r) 125,000), was partially purified from lobster muscle; enzyme activity coeluted with a 60-kDa polypeptide using anion-exchange chromatography. The 60-kDa protein reacted with a polyclonal antibody raised against a 20-amino acid peptide sequence found around the catalytic cysteine residue of mu- and m-calpains, but not with antibodies raised against other regions of mu- or m-calpain or with the anti-CDP IIb antibody. These results suggest that (1) the CDP IIb is the homolog of Drosophila calpain in crustaceans and (2) the active site regions of CDP IIa and mu- and m-calpains are similar.

Amino Acid Sequence↗
PubMed2002

Activity profile of calpains I and II in chronically infarcted rat myocardium--influence of the calpain inhibitor CAL 9961.

1. The calpains have been proposed to be activated following cardiac ischaemia and to contribute to myocyte damage after myocardial infarction (MI). In this study, the activity of calpains I and II in the infarcted and non-infarcted rat myocardium and the action of the selective calpain inhibitor, CAL 9961, has been investigated. 2. MI was induced by permanent ligation of the left coronary artery. One, 3, 7 and 14 days post MI, the enzymes calpain I and II were separated from homogenates of the interventricular septum (IS) and left ventricular free wall (LVFW) by chromatography on DEAE-Sepharose. The activity of the calpains was measured in sham-operated and MI animals chronically treated with placebo or CAL 9961 (15 mg kg(-1) d(-1) s.c.) in a synthetic substrate assay. Treatment was started 3 days before MI induction. 3. Calpain I activity reached highest values in IS 14 days post MI, whereas maximum activity of calpain II was measured in LVFW 3 days post MI. In experiments in vitro, CAL 9961 completely inhibited both calpains. In vivo, chronic treatment of MI animals with CAL 9961 partially prevented the increase in calpain I activity in IS and reduced calpain II activity in LVFW to sham levels. 4. Our findings demonstrate that calpains I and II are activated after MI, however, both enzymes differ in their regional and temporal activation within the infarcted myocardium. Chronic inhibition of these enzymes with CAL 9961 might limit the calpain-induced myocardial damage and preserve cardiac structural integrity post MI.

Animals↗
PubMed2002

In vitro antioxidant properties of calpain inhibitors: leupeptin and calpain inhibitor-1.

The aim of this study was 1/ to assess the efficacy of calpain inhibitors: leupeptin and calpain inhibitor-1, to inhibit calpain activity in vitro, 2/ to measure the scavenging abilities of these compounds against free radicals. The efficacy of calpain inhibitors to block calpain activity was tested with azocasein as a substrate for calpain. Leupeptin and calpain inhibitor-1 inhibited calpain activity in the same range of concentrations, the IC50 being 0.14 microM and 0.09 microM, respectively. We measured the antioxidant properties of leupeptin and calpain inhibitor-1 using the allophycocyans assay after identification offree radical species produced by the complex H2O2 + Cu(++). Electron paramagnetic resonance (EPR) spin-trapping studies performed by using DMPO showed that a quartet signal (hyperfine couplings aN = aH = 14.9 G) arisen from DMPO-OH was formed. We found a correlation between leupeptin concentration and oxygen radical absorbance capacity (ORAC) values (r2 = 0.975) indicating an in vivo antioxidant capacity. In contrast, calpain inhibitor-1 showed no protection. In conclusion, our findings indicate that leupeptin and calpain inhibitor-1 are equipotent inhibitors on calpain activity but exhibit diffrent antioxidant efficacy.

Antioxidants↗
PubMed2003

Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors.

Spinal cord injury (SCI) evokes an increase in intracellular free Ca(2+) level resulting in activation of calpain, a Ca(2+)-dependent cysteine protease, which cleaves many cytoskeletal and myelin proteins. Calpain is widely expressed in the central nervous system (CNS) and regulated by calpastatin, an endogenous calpain-specific inhibitor. Calpastatin degraded by overactivation of calpain after SCI may lose its regulatory efficiency. Evidence accumulated over the years indicates that uncontrolled calpain activity mediates the degradation of many cytoskeletal and membrane proteins in the course of neuronal death and contributes to the pathophysiology of SCI. Cleavage of the key cytoskeletal and membrane proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells leading to cell death. Calpain in conjunction with caspases, most notably caspase-3, can cause apoptosis of the CNS cells following trauma. Aberrant Ca(2+) homeostasis following SCI inevitably activates calpain, which has been shown to play a crucial role in the pathophysiology of SCI. Therefore, calpain appears to be a potential therapeutic target in SCI. Substantial research effort has been focused upon the development of highly specific inhibitors of calpain and caspase-3 for therapeutic applications. Administration of cell permeable and specific inhibitors of calpain and caspase-3 in experimental animal models of SCI has provided significant neuroprotection, raising the hope that humans suffering from SCI may be treated with these inhibitors in the near future.

Animals↗
PubMed1996

mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury.

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent mu-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Analysis of Variance↗
PubMed2006

Oestrogen receptor subtype-specific repression of calpain expression and calpain enzymatic activity in neuronal cells--implications for neuroprotection against Ca-mediated excitotoxicity.

Calpains represent a superfamily of Ca2+-activated cysteine-proteases, which are important mediators of apoptosis and necrosis. In the brain, m-calpain and micro-calpain, the two ubiquitous calpain-isoforms, are strongly activated in neurones after an excitotoxic Ca2+ influx occurring, for example, during cerebral ischemia. Because oestrogen and its receptors (ERalpha/ERbeta) can exert neuroprotective activity, we investigated their influence on expression of calpains and their endogenous inhibitor, calpastatin. We found that ectopic expression of ERalpha in human neuroblastoma SK-N-MC cells led to a ligand-independent constitutive down-regulation of m-calpain accompanied by an up-regulation of micro-calpain expression. Up-regulation of micro-calpain was reversed in the presence of oestrogen, which, in turn, could be blocked by co-treatment with the oestrogen-receptor antagonist ICI 182,780. Expression of calpastatin was not altered, either in the absence or in the presence of oestrogen. Additional studies revealed that ERalpha-expressing cells exhibited decreased calpain enzymatic activity and increased survival when cells were exposed to the Ca2+ ionophore, ionomycin. Since all investigated effects could be observed exclusively in the presence of ERalpha, but not ERbeta, and since the effects are reduced when ERalpha and ERbeta are co-expressed, our data suggest a novel ER subtype-specific neuroprotective action by repressing calpain expression and calpain activity under conditions of a massive Ca2+ influx.

Brain↗