Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.
Calcium/pharmacology ; Calpain/metabolism* ; Calpain/antagonists & inhibitors* ; Cell Differentiation ; Dose-Response Relationship, Drug ; Epidermis/metabolism ; Human ; In Vitro ; Keratinocytes/metabolism ; Keratinocytes/enzymology ; Protease Inhibitors/pharmacology ; Protein-Glutamine gamma-Glutamyltransferase/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/antagonists & inhibitors* ; Tissue Culture

Calpains, calcium-activated cysteine proteases with a neutral pH optimum, lead to degration of cystoskeletion and structural protein, and delayed neuronal death. The activation of calpains contribute to apoptosis. Calpain inhibitors provide a novel and potential treatment for cerebral ischemia due to improvement of cerebral infarct and ischemia.
Animals ; Apoptosis ; drug effects ; Brain Ischemia ; drug therapy ; metabolism ; Calcium-Binding Proteins ; therapeutic use ; Calpain ; antagonists & inhibitors ; metabolism ; physiology ; Humans

Primary neuronal culture is a powerful tool to study neuronal development, aging, and degeneration. However, cultured neurons show signs of cell death after 2 or 3 weeks. Although the mechanism underlying this phenomenon has not been elucidated, several preventive methods have been identified. Here we show that the neuronal loss in primary cortical culture involves calpain activation and subsequent neuronal cell death. Neuronal loss during cultivation showed destruction of neurites and synapses, and a decrease in neuron numbers. micro-Calpain and micro-calpain were initially activated and accumulated by increased RNA expression. This neuronal death exhibited neurodegenerative features, such as conversion of p35 to p25, which is important in the developmental process and in the pathogenesis of Alzheimer's disease. But, postnatal and aged rat cortex did not show calpain activation and prolonged processing of p35 to p25, in contrast to the long-term culture of cortical neurons. In addition, the inhibition of calpains by ALLM or ALLN blocked the conversion of p35 to p25, indicating that the calpain activity is essential for the neurodegenerative features of cell death. Taken together, this study shows that the neuronal loss in primary cortical cultures involves neurodegeneration-like cell death through the activation of calpains and the subsequent processing of p35 to p25, but not developmental apoptosis or aging. Our results suggest that the long term primary culture of cortical neurons represent a valuable model of neurodegeneration, such as Alzheimer's disease.
Transcription, Genetic/genetics ; Time Factors ; Rats ; Phosphotransferases/*metabolism ; Neurons/*cytology/*metabolism ; Cells, Cultured ; Cell Shape ; Caspases/antagonists & inhibitors/metabolism ; Calpain/antagonists & inhibitors/genetics/*metabolism ; *Apoptosis ; Animals

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and micro-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and micro-calpain may be involved in colon epithelial cell death induced by E. histolytica.
Calpain/antagonists & inhibitors/genetics/*metabolism ; *Cell Death ; Cell Line ; Cell Survival/drug effects ; Dipeptides/metabolism ; Entamoeba histolytica/*pathogenicity ; Epithelial Cells/*parasitology ; Gene Knockdown Techniques ; Humans

BACKGROUNDAtrial fibrillation (AF) is accompanied by atrial structural remodeling. Calpain activity is induced during AF. To test a causal relationship between calpain activation and atrial structural changes, N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, was utilized in a canine AF model.

METHODSFifteen dogs were randomly divided into 3 groups: sham-operated group, control group and calpain inhibitor group; each with 5 dogs. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute for 3 weeks. ALLM was administered at a dosage of 1.0 mg x kg(-1) x d(-1) in the calpain inhibitor group. Three weeks later, the proteolysis, protein expression of TnT and myosin, calpain I localization and expression and structural changes were examined in left atrial free walls, right atrial free walls and the interatrial septum respectively. Atrial size and contractile function were also measured by echocardiography.

RESULTSLong-term rapid atrial pacing induced marked structural changes such as enlarged atrial volume, myolysis, degradation of TnT and myosin, accumulation of glycogen and changes in mitochondrial shape and size, which were paralleled by an increase in calpain activity. The positive correlation between calpain activity and the degree of myolysis (r(s) = 0.90 961, P < 0.0001) was demonstrated. In addition to structural abnormalities, pacing-induced atrial contractile dysfunction was observed in this study. The pacing-induced atrial structural alterations and loss of contractility were partially prevented by the calpain inhibitor ALLM.

CONCLUSIONSActivation of calpain represents key features in the progression towards overt structural remodeling. Calpain inhibitor, ALLM, suppressed the increased calpain activity and reversed structural remodeling caused by sustained atrial fibrillation in the present model. Calpain inhibition may therefore provide a possibility for therapeutic intervention in AF.

Animals ; Atrial Fibrillation ; pathology ; Calpain ; antagonists & inhibitors ; Cysteine Proteinase Inhibitors ; pharmacology ; Disease Models, Animal ; Dogs ; Heart Atria ; pathology ; ultrastructure ; Myosins ; analysis ; Troponin T ; analysis

OBJECTIVETo investigate the changes in the functional activity of glycogen synthase kinase-3 (GSK-3) in the hepatic tissue after endotoxin (lipopolysaccharide, LPS) tolerance and explore the effects of LPS-induced GSK-3 inhibition on glycogen metabolism in the liver.

METHODSMale SD rats were randomly divided into normal control, endotoxin pretreatment and GSK-3 inhibitor (lithium chloride) groups with corresponding pretreatments prior to a large dose of LPS challenge (10 mg/kg) to induce liver injury. Glycogen deposition and content in the hepatic tissue was detected using periodic acid-Schiff (PAS) staining and a glycogen quantification kit, respectively. Western blotting was performed for semi-quantitative analysis of protein level and inhibitory phosphorylation of GSK-3, and a Coomassie brilliant blue G-250-based colorimetric assay was used to detect calpain activity in the liver.

RESULTSGlycogen content in the liver decreased significantly after LPS challenge in all the 3 groups (P<0.05) but showed no significant difference among the groups (P>0.05). Both LPS and lithium chloride pretreatments caused a significant increase of liver glycogen content (P<0.05). LPS pretreatment induced inhibitory phosphorylation of GSK-3β (P<0.05) and partial cleavage of GSK-3α but did not affect the expression of GSK-3 protein (P>0.05). Large-dose LPS challenge significantly increased the activity of calpain in the liver tissue (P<0.05) to a comparable level in the 3 groups (P>0.05).

CONCLUSIONEndotoxin pretreatment induces inhibitory phosphorylation of GSK-3β and partial cleavage of GSK-3α and promotes the deposition of liver glycogen but does not affect the activity of calpain, which may contribute to an increased glycogen reserve for energy supply in the event of large-dose LPS challenge.

Animals ; Calpain ; metabolism ; Glycogen ; metabolism ; Glycogen Synthase Kinase 3 ; antagonists & inhibitors ; metabolism ; Lipopolysaccharides ; adverse effects ; Lithium Chloride ; pharmacology ; Liver ; drug effects ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the relation of the structural remodeling processes and activation of calpain I.

METHODSFifteen dogs were randomly divided into three groups. The dogs in pacing group (n=5) and inhibitor group (n=5) were subjected to 3 weeks of rapid atrial pacing at 600 beats/min, control dogs (n=5) were in sham-operated group. The dogs in inhibitor group were administered intravenous N-Acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, and in pacing group and sham-operated group were administered intravenous DMSO. The activity of calpain I was measured by hydrolyzing Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin. The ultrastructure of atrium was examined by light and electron microscopy. TnT expression was assessed by Western blot. Echocardiography examination was performed in all the three groups.

RESULTSCalpain I activity was significantly increased in pacing group (2.3-fold, P<0.01), and decreased in inhibitor group (1.1-fold, P>0.05), compared to sham-operated group respectively. The percentages of myolysis were (76.7 +/- 5.9)% and (20.8 +/- 8.1)% in pacing group and inhibitor group respectively (P<0.01). TnT expression decreased in the rapid pacing-induced persistent atrial fibrillation, and these effects were inhibited by calpain I inhibitor ALLM. The area and volume of left atrium tended to increase after 3 weeks ALLM treatment in inhibitor group, but the change was not as prominent as in pacing group (P<0.05).

CONCLUSIONSALLM can decrease calpain I activity, and prevent canine atrial cardiomyocyte structural remodeling during atrial fibrillation. This study provided a capacity of atrial cardiomyocyte protection.

Animals ; Atrial Fibrillation ; metabolism ; physiopathology ; Atrial Function, Left ; Calpain ; antagonists & inhibitors ; metabolism ; Cardiac Pacing, Artificial ; Disease Models, Animal ; Dogs ; Heart Atria ; ultrastructure ; Myocardium ; metabolism ; Troponin T ; metabolism

OBJECTIVETo study the effect of leptin on the expression of calcium-activated neutral protease 1 (calpain-1) and B cell lymphoma-2 (Bcl-2) and apoptosis in the myocardial tissue of neonatal rats after asphyxia.

METHODSA total of 48 neonatal rats were randomly and equally divided into normal control group, asphyxia group, leptin treatment groups, and calpain-1 inhibitor (CAI-1) group. The neonatal rat model of asphyxia under normal atmospheric condition was established in all groups except the control group. For the leptin treatment groups, rats received 20, 80, and 160 μg/kg leptin by intraperitoneal injection immediately after model establishment, respectively. For the CAI-1 group, rats received 10 mg/kg CAI-1 by intraperitoneal injection immediately after model establishment. For all the groups, the myocardial tissue was collected at 2 hours after model establishment. Immunohistochemistry was used to measure the expression of calpain-1 and Bcl-2. The TUNEL method was used to evaluate apoptosis of myocardial cells.

RESULTSThe expression of calpain-1 and Bcl-2 and apoptosis index (AI) were significantly higher in the asphyxia group than in the normal control group (P˂0.05). The leptin treatment groups and the CAI-1 group had significantly lower expression of calpain-1, significantly lower AI, and significantly higher expression of Bcl-2 than the asphyxia group (P˂0.05). The CAI-1 group had the largest changes in all the indices compared with the asphyxia group. However, there were no significant differences in all indices between the 160 μg/kg leptin treatment group and the CAI-1 group. After asphyxia, the expression of calpain-1 was positively correlated with AI, while the expression of Bcl-2 was negatively correlated with AI and the expression of calpain-1 (P˂0.05).

CONCLUSIONSLeptin reduces apoptosis of myocardial cells in asphyxiated neonatal rats by the inhibition of calpain-1 activation and upregulation of Bcl-2 expression.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Asphyxia Neonatorum ; metabolism ; pathology ; Calpain ; analysis ; antagonists & inhibitors ; Leptin ; pharmacology ; Myocardium ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.

METHODSWashed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer.

RESULTSAfter PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor.

CONCLUSIONPKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.

Blood Platelets ; drug effects ; Calpain ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; antagonists & inhibitors ; Flow Cytometry ; Humans ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Platelet Glycoprotein GPIb-IX Complex ; biosynthesis

BACKGROUNDIn addition to neurons, all components of the neurovascular unit (NVU), such as glial, endothelial, and basal membranes, are destroyed during traumatic brain injury (TBI). Previous studies have shown that excessive stimulation of calpain is crucial for cerebral injury after traumatic insult. The objective of this study was to investigate whether calpain activation participated in NVU disruption and edema formation in a mouse model of controlled cortical impact (CCI).

METHODSOne hundred and eight mice were divided into three groups: the sham group, the control group, and the MDL28170 group. MDL28170 (20 mg/kg), an efficient calpain inhibitor, was administered intraperitoneally at 5 min, 3 h, and 6 h after experimental CCI. We then measured neurobehavioral deficits, calpain activity, inflammatory mediator levels, blood-brain barrier (BBB) disruption, and NVU deficits using electron microscopy and histopathological analysis at 6 h and 24 h after CCI.

RESULTSThe MDL28170 treatment significantly reduced the extent of both cerebral contusion (MDL28170 vs. vehicle group, 16.90 ± 1.01 mm΃ and 17.20 ± 1.17 mm΃ vs. 9.30 ± 1.05 mm΃ and 9.90 ± 1.17 mm΃, both P < 0.001) and edema (MDL28170 vs. vehicle group, 80.76 ± 1.25% and 82.00 ± 1.84% vs. 82.55 ± 1.32% and 83.64 ± 1.25%, both P < 0.05), improved neurological scores (MDL28170 vs. vehicle group, 7.50 ± 0.45 and 6.33 ± 0.38 vs. 12.33 ± 0.48 and 11.67 ± 0.48, both P < 0.001), and attenuated NVU damage resulting (including tight junction (TJ), basement membrane, BBB, and neuron) from CCI at 6 h and 24 h. Moreover, MDL28170 markedly downregulated nuclear factor-κB-related inflammation (tumor necrosis factor-α [TNF-α]: MDL28170 vs. vehicle group, 1.15 ± 0.07 and 1.62 ± 0.08 vs. 1.59 ± 0.10 and 2.18 ± 0.10, both P < 0.001; inducible nitric oxide synthase: MDL28170 vs. vehicle group, 4.51 ± 0.23 vs. 6.23 ± 0.12, P < 0.001 at 24 h; intracellular adhesion molecule-1: MDL28170 vs. vehicle group, 1.45 ± 0.13 vs. 1.70 ± 0.12, P < 0.01 at 24 h) and lessened both myeloperoxidase activity (MDL28170 vs. vehicle group, 0.016 ± 0.001 and 0.016 ± 0.001 vs. 0.024 ± 0.001 and 0.023 ± 0.001, P < 0.001 and 0.01, respectively) and matrix metalloproteinase-9 (MMP-9) levels (MDL28170 vs. vehicle group, 0.87 ± 0.13 and 1.10 ± 0.10 vs. 1.17 ± 0.13 and 1.25 ± 0.12, P < 0.001 and 0.05, respectively) at 6 h and 24 h after CCI.

CONCLUSIONSThese findings demonstrate that MDL28170 can protect the structure of the NVU by inhibiting the inflammatory cascade, reducing the expression of MMP-9, and supporting the integrity of TJ during acute TBI.

Animals ; Brain Injuries, Traumatic ; drug therapy ; metabolism ; Calpain ; antagonists & inhibitors ; metabolism ; Dipeptides ; therapeutic use ; Disease Models, Animal ; Glycoproteins ; therapeutic use ; Inflammation ; drug therapy ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; metabolism ; Peroxidase ; antagonists & inhibitors ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism