calpain and calpain-inhibitor-2

B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common hematological disorder among middle-aged/elderly people in the Western countries. We have shown earlier that B-CLL cells exhibit elevated total amount and available activity of µ-calpain, belonging to a family of ubiquitous, strongly Ca-dependent proteases, involved in the control of proliferation and apoptosis. In this study we attempted to estimate a potential clinical value of μ-calpain in relation to B-CLL clinical staging in patients with extremely high lymphocytosis and studied the molecular mechanisms associating calpain activity with clinical progress of the disease. We observed significant correlations between the amounts of intracellular μ-calpain and clinical staging of the disease, with RAI stage 1 corresponding to the highest calpain amounts in the leukemic cells. There was also a positive, statistically significant correlation between the amount of μ-calpain and phosphorylated (p)ZAP-70 in B-CLL lymphocytes. Calpain activity in the B-CLL cells is associated with decreased activities of pro-apoptotic caspases -3 and -9, and reciprocally with an increased amount of anti-apoptotic Bcl-2. Together, all of these findings make calpain activity in B-CLL cells a promising target modifying the properties of these cells and facilitating therapy. Finally, the proportion of CD19+ B cells with elevated μ-calpain and pZap-70 was markedly reduced in patients after successful therapy.

Topics: Aged; Aged, 80 and over; Apoptosis; B-Lymphocytes; Calpain; Case-Control Studies; Caspase 3; Caspase 9; Cells, Cultured; Disease Progression; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Staging; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; ZAP-70 Protein-Tyrosine Kinase

Traumatic Brain Injury (TBI) is a major cause of death and disability worldwide. The calcium-dependent protease, calpain, has been shown to be involved in TBI-induced neuronal death. However, whereas various calpain inhibitors have been tested in several animal models of TBI, there has not been any clinical trial testing the efficacy of calpain inhibitors in human TBI. One important reason for this could be the lack of knowledge regarding the differential functions of the two major calpain isoforms in the brain, calpain-1 and calpain-2. In this study, we used the controlled cortical impact (CCI) model in mice to test the roles of calpain-1 and calpain-2 in TBI-induced neuronal death. Immunohistochemistry (IHC) with calpain activity markers performed at different time-points after CCI in wild-type and calpain-1 knock-out (KO) mice showed that calpain-1 was activated early in cortical areas surrounding the impact, within 0-8 h after CCI, whereas calpain-2 activation was delayed and was predominant during 8-72 h after CCI. Calpain-1 KO enhanced cell death, whereas calpain-2 activity correlated with the extent of cell death, suggesting that calpain-1 activation suppresses and calpain-2 activation promotes cell death following TBI. Systemic injection(s) of a calpain-2 selective inhibitor, NA101, at 1 h or 4 h after CCI significantly reduced calpain-2 activity and cell death around the impact site, reduced the lesion volume, and promoted motor and learning function recovery after TBI. Our data indicate that calpain-1 activity is neuroprotective and calpain-2 activity is neurodegenerative after TBI, and that a selective calpain-2 inhibitor can reduce TBI-induced cell death.

Topics: Animals; Apoptosis; Brain Injuries, Traumatic; Calpain; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Neuroprotective Agents; Oligopeptides; Recovery of Function

Overexposure to manganese has been known to promote alpha-synuclein oligomerization and enhance cellular toxicity. However, the exact mechanism of Mn-induced alpha-synuclein oligomerization is unclear. To explore whether alpha-synuclein oligomerization was associated with the cleavage of alpha-synuclein by calpain, we made a rat brain slice model of manganism and pretreated slices with calpain inhibitor II, a cell-permeable peptide that restricts the activity of calpain. After slices were treated with 400 μM Mn for 24 h, there were significant increases in the percentage of apoptotic cells, lactate dehydrogenase release, intracellular [Ca2+]i, calpain activity, and the mRNA and protein expression of calpain 1 and alpha-synuclein. Moreover, the number of C- and N-terminal fragments of alpha-synuclein and the amount of alpha-synuclein oligomerization also increased. These results also showed that calpain inhibitor II pretreatment could reduce Mn-induced nerve cell injury and alpha-synuclein oligomerization. Additionally, there was a significant decrease in the number of C- and N-terminal fragments of alpha-synuclein in calpain inhibitor II-pretreated slices. These findings revealed that Mn induced the cleavage of alpha-synuclein protein via overactivation of calpain and subsequent alpha-synuclein oligomerization in cultured slices. Moreover, the cleavage of alpha-synuclein by calpain 1 is an important signaling event in Mn-induced alpha-synuclein oligomerization.

Topics: alpha-Synuclein; Animals; Calpain; Gene Expression; Manganese; Oligopeptides; Protein Multimerization; Proteolysis; Rats, Wistar; Tissue Culture Techniques

Childhood acute lymphoblastic leukemia (ALL) blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS) composed of cytoplasmic proteases--calpains--performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor--calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher μ-calpain (CAPN1) gene transcription, protein amounts and activity (but not those of m-calpain), with calpastatin amount and transcription of its gene (CAST) greatly varying were observed in CD19(+) ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk) displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL.

Topics: Adolescent; Age Factors; Apoptosis; B-Lymphocytes; Blast Crisis; Calpain; Cell Proliferation; Child; Child, Preschool; Female; Humans; Infant; Male; Neoplasm Proteins; Oligopeptides; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma

The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological modulation of calpain activity prevents spheroid formation and causes disassembly of preexisting spheroids into single cells, thereby providing novel strategies for improving suspension culture conditions for human pluripotent stem cells in the future.

Topics: beta Catenin; Cadherins; Calpain; Cell Adhesion; Cell Culture Techniques; Cells, Cultured; Cysteine Proteinase Inhibitors; Gene Expression Regulation; Glycoproteins; High-Throughput Nucleotide Sequencing; Humans; Isotope Labeling; Oligopeptides; Pluripotent Stem Cells; Proteomics; Sequence Analysis, RNA; Wnt Signaling Pathway

Matrix metalloproteinase (MMP)-2 is a zinc-dependent endopeptidase which, alongside its known extracellular actions, plays fundamental roles in oxidative stress-induced injury to the heart. Intracellular cleavage targets of MMP-2 selectively mediating this injury include the sarcomeric proteins troponin I, myosin light chain-1 and titin; some of these are also targeted by calpains. In myocardial ischemia and reperfusion injury, inhibitors of MMP-2 and some calpain inhibitors were shown to improve the recovery of contractile function. We hypothesized that the protective effects of calpain inhibitors may be due in part to their ability to inhibit MMP-2. Four calpain inhibitors (calpain inhibitor III, ALLM, ALLN, and PD-150606) were tested for their ability to inhibit MMP-2 in comparison to the selective MMP inhibitor ONO-4817. At 100 μM, all calpain inhibitors, except ALLM, showed significant inhibition of MMP-2 gelatinolytic activity. When assessed by the troponin I proteolysis assay, both ALLN and PD-150606, but neither ALLM nor calpain inhibitor III (at 20 μM), significantly inhibited MMP-2 activity. Using a fluorogenic MMP substrate peptide OmniMMP in a kinetic assay the rank order of IC(50) values against MMP-2 were: PD-150606

Topics: Acrylates; Calpain; Catalysis; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Gelatin; Humans; Inhibitory Concentration 50; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Oligopeptides; Phenyl Ethers; Troponin I

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

Topics: Animals; Autolysis; Blotting, Western; Calpain; Cell Fractionation; Cytosol; Disease Models, Animal; Dogs; Electrophoresis, Polyacrylamide Gel; Intracellular Membranes; Leupeptins; Male; Muscle, Skeletal; Myocardial Reperfusion Injury; Myocardium; Oligopeptides; Rabbits; Rats; Rats, Sprague-Dawley

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O(2). A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2alpha is an orthologue of HIF-1alpha, we examined the effects of IH on HIF-2alpha, the O(2)-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1alpha, HIF-2alpha was down-regulated by IH. Similar down-regulation of HIF-2alpha was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2alpha whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2alpha degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2alpha prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2alpha degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2alpha via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.

Topics: Animals; Apnea; Autonomic Nervous System; Basic Helix-Loop-Helix Transcription Factors; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Down-Regulation; Enzyme Activation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Oligopeptides; Oxidative Stress; PC12 Cells; Protein Binding; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

The objective of this study was to examine the potential of arsenate to induce cardiomyocyte cell death and to explore the cellular mechanisms of arsenate toxicity. Isolated cardiomyocytes in culture from embryonic chick hearts were treated with a pentavalent arsenic species (H3AsO4) or arsenate. Arsenate produced a significant (P < 0.01) concentration-dependent increase in cell death with an EC50 about 1 mM. Cardiomyocytes manifested a loss of actin structure, reduced size, and damaged nuclei. Creatine 0.1-100 uM did not significantly modify arsenate-induced cell death. In contrast, verapamil, 0.01-1 uM, produced a significant concentration-dependent accentuation of arsenate-induced cell death. The effect of verapamil was evident at low concentrations of arsenate, which produced only a small increase in cell death, and at high concentrations of arsenate, which induced a large amount of cell death. Verapamil alone did not alter cardiomyocyte cell death. By comparison, calpain inhibitor II did not modify arsenate-induced cardiomyocyte cell death. These data suggest that cardiomyocytes are vulnerable to the effects of verapamil to increase the cellular toxicity of arsenate. Two potential cellular mechanisms of arsenate toxicity, however, are likely not involved in arsenate toxicity namely calpain activation and reduction of creatine phosphate production.

Topics: Animals; Arsenates; Calpain; Cell Death; Chick Embryo; Creatine; Dose-Response Relationship, Drug; Myocytes, Cardiac; Oligopeptides; Verapamil

Topics: Animals; Apoptosis; Atrial Fibrillation; Calpain; Caspase 7; Dogs; Myocytes, Cardiac; Oligopeptides

Degenerative retinopathies, including age-related macular degeneration, diabetic retinopathy, and hereditary retinal disorders--major causes of world blindness--are potentially treatable by using low-molecular weight neuroprotective, antiapoptotic, or antineovascular drugs. These agents are, however, not in current systemic use owing to, among other factors, their inability to passively diffuse across the microvasculature of the retina because of the presence of the inner blood-retina barrier (iBRB). Moreover, preclinical assessment of the efficacies of new formulations in the treatment of such conditions is similarly compromised. We describe here an experimental process for RNAi-mediated, size-selective, transient, and reversible modulation of the iBRB in mice to molecules up to 800 Da by suppression of transcripts encoding claudin-5, a protein component of the tight junctions of the inner retinal vasculature. MRI produced no evidence indicative of brain or retinal edema, and the process resulted in minimal disturbance of global transcriptional patterns analyzed in neuronal tissue. We show that visual function can be improved in IMPDH1(-/-) mice, a model of autosomal recessive retinitis pigmentosa, and that the rate of photoreceptor cell death can be reduced in a model of light-induced retinal degeneration by systemic drug delivery after reversible barrier opening. These findings provide a platform for high-throughput drug screening in models of retinal degeneration, and they ultimately could result in the development of a novel "humanized" approach to therapy for conditions with little or no current forms of treatment.

Topics: Animals; Blood-Retinal Barrier; Calpain; Claudin-5; Cysteine Proteinase Inhibitors; Disease Models, Animal; Drug Delivery Systems; Electroretinography; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Magnetic Resonance Imaging; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oligopeptides; Retina; Retinitis Pigmentosa; RNA Interference; RNA, Small Interfering

This study was designed to evaluate the effects of a calpain inhibitor on cardiac muscle apoptosis in rapid pacing canine atrial fibrillation (AF) models.. Twenty one dogs were divided into three groups: a sham operation group, a control AF group and a calpain inhibitor group. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute. N-Acetyl-Leu-Leu-Met (1.0 mg/kg/day) was administered in the calpain inhibitor group for three weeks. The activity of calpain I and cardiomyocyte apoptosis were measured by fluorometry and TUNEL assay, respectively. Protein expression of caspase-3 was detected by Western blot. The localizations of caspase-3, caspase-8, bcl-2 and ARC were assessed by immunohistochemistry.. In comparison to the sham operation group, the activity of calpain I was significantly increased in the control AF group (2.3 fold, p < 0.001), and decreased in the calpain inhibitor group (1.1 fold, p < 0.005). The calpain activity correlated with the apoptosis index (r = 0.9, p < 0.05). The apoptosis index was 1.0 +/- 0.2%, 11.8 +/- 6.8% and 3.5 +/- 2.1% in the sham operation group, control AF group and calpain inhibitor group, respectively. In the sham operation group, control AF group and calpain inhibitor group, the expressions of caspase-3 (13.0 +/- 1.9%, 52.8 +/- 4.3% and 33.6 +/- 3.7%), caspase-8 (40.1 +/- 5.3%, 92.6 +/- 6.5% and 55.3 +/- 5.9%), bcl-2 (65.8 +/- 6.1%, 52.0 +/- 5.7% and 69.9 +/- 5.3%) and ARC (70.2 +/- 8.6%, 68.8 +/- 7.3% and 81.5 +/- 8.8%) were calculated as immunohistochemical indexes, respectively.. The calpain inhibitor N-Acetyl-Leu-Leu-Met attenuated apoptosis through a complicated network of apoptosis-related proteins, which may result in improvement of structural remodeling in atrial fibrillation.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Atrial Fibrillation; Blotting, Western; Body Weight; Calpain; Caspase 3; Dogs; Immunohistochemistry; In Situ Nick-End Labeling; Myocardial Contraction; Myocytes, Cardiac; Oligopeptides; Organ Size; Protease Inhibitors; RNA, Messenger

We investigated the response of alphaB-crystallin to oxidative stress and calpain inhibition in an attempt to elucidate the signalling pathways mediating its phosphorylation. Given the high expression levels of alphaB-crystallin in cardiac muscle one can evaluate the significance of its participation in preservation of homeostasis under adverse conditions. H9c2 cardiac myoblasts were used as our experimental model since their response reflects the signal transduction pathways activated by stress conditions in the myocardium. Thus, in H9c2 cells treated with H2O2 the mechanism regulating alphaB-crystallin phosphorylation was found to involve p38-MAPK/MSK1 as well as intracellular free calcium levels. Our immunocytochemical experiments demonstrated phosphorylated alphaB-crystallin to be co-localized with tubulin, potentially preserving cytoskeletal architecture under these interventions. In H9c2 cells treated with calpain inhibitors (ALLN, ALLM) alphaB-crystallin exhibited a p38-MAPK- and [Ca 2+](i)-dependent phosphorylation pattern since the latter was ablated in the presence of the selective p38-MAPK inhibitor SB203580 and calcium chelator BAPTA-AM. Calpain activity repression ultimately led to apoptosis confirmed by PARP fragmentation and chromatin condensation. However, the apoptotic pathway activated by ALLM and ALLN differed, underlying the diverse transduction mechanisms stimulated. In addition to this, an anti-apoptotic role for phospho-alphaB-crystallin was verified by confirmation of its interaction with pro-caspase 3, hindering its cleavage and subsequent activation. Collectively, our findings underline alphaB-crystallin crucial role as a participant of cardiac cells early response to stressful stimuli compromising their survival.

Topics: alpha-Crystallin B Chain; Animals; Apoptosis; Calcium Signaling; Calpain; Caspase 3; Cell Line; Cell Nucleus; Cytochromes c; Egtazic Acid; Hydrogen Peroxide; Leupeptins; Myocytes, Cardiac; Oligopeptides; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Phosphoserine; Poly(ADP-ribose) Polymerases; Protein Transport; Rats; Ribosomal Protein S6 Kinases, 90-kDa; Time Factors; Tubulin

Neovascularization is an essential process in tumor development, it is conceivable that anti-angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho-[1,2-b]pyran-5,6-dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti-cancer and anti-viral effects. Whether beta-lapachone can induce endothelial cell death or has an anti-angiogenic effect is still an enigma. We investigated the in vitro effect of beta-lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during beta-lapachone-induced endothelial cell death; (2) co-treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD-fmk, provided significant protection against apoptosis by preventing the beta-lapachone-induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the beta-lapachone-induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by beta-lapachone, but not the anti-angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of beta-lapachone on human endothelial cells and suggest that beta-lapachone may have potential as an anti-angiogenic drug.

Topics: Angiogenesis Inhibitors; Apoptosis; Arginine; Calcium; Calpain; Caspases; Cell Line; Cell Survival; Chelating Agents; Cyclic GMP; Dose-Response Relationship, Drug; Egtazic Acid; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Humans; Leupeptins; Membrane Potential, Mitochondrial; Naphthoquinones; Neovascularization, Physiologic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Oligopeptides; Signal Transduction; Time Factors

To test the causal relationship between calpain activation and atrial structural changes during atrial fibrillation (AF).. The tip of a spiral mono-polar pacing lead was fixed to the right atrial appendages of 15 dogs randomly divided into 3 equal groups: calpain inhibitor group, undergoing continuous pacing with the impulse of 600 beats/min for 3 weeks and intravenous injection of N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor for 3 weeks; control group, undergoing continuous pacing and intravenous injection of dimethyl sulfoxide (DMSO; and sham operation group, given DMSO injection without pacing. Ultrasonography was used to observe the changes of the structures of left atrium and left atrial appendage and the heart function as well. Specimens of atrial muscles were obtained. Calpain 1 activity was detected by Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin method. HE staining was conducted to observe the myolysis. Western blotting was used to detect the protein expression of troponin I (TnI) and myosin.. The left atrial ejection fraction (LAEF) of the ALLM group was (41 +/- 6)%, significantly higher than that of the control group [(34 +/- 9)%, P < 0.05]. The left atrial appendage ejection fraction (LAAEF) of the ALLM group was (41 +/- 6)%, significantly higher than that of the control group [(35 +/- 6)%, P < 0.05]. Myolysis was extensive in the control group [(71.5 +/- 10.2)%], relatively rare in the ALLM group [(12.3 +/- 16.5)%], and was not seen in the sham operation group, with significantly differences among the 3 groups (all P < 0.01). The calpain 1 activity was positively correlated with the degree of myolysis (r(s) = 0.90 961, P < 0.01). The TnI level of the control group was (43 +/- 12)% that of the sham operation group (P = 0.001), the TnI level of the ALLM group was (51 +/- 11)% that of the sham operation group (P = 0.002) and was significant higher than that of the control group (P = 0.01). The level of myosin of the control group was (51 +/- 11)% that of the sham operation group (P = 0.002), and that of the ALLM group was (149 +/- 33)% that of the control group (P = 0.005).. Activation of and upregulation of expression of calpain participate in the structural remodeling of left atrial cardiac muscle and contractile dysfunction. Calpain inhibitor suppresses the increased calpain activity and reverses the structural remodeling of sustained atrial fibrillation. Calpain inhibition may therefore provide a possibility for therapeutic intervention in AF.

Topics: Animals; Atrial Fibrillation; Blotting, Western; Calpain; Disease Models, Animal; Dogs; Heart; Injections, Intravenous; Myocardial Contraction; Myocardium; Myosins; Oligopeptides; Random Allocation

Treatment of neurons with okadaic acid, a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death, and to create a research model of Alzheimer's disease. Amyloid precursor protein (APP) is the precursor protein of the beta-amyloid peptide that accumulates in extracellular plaques in Alzheimer's disease. Several studies have shown that mint-1 (munc18-interacting protein 1) and mint-2 bind to the YENPTY motif in the cytoplasmic domain of APP and inhibit processing of APP to beta-amyloid peptide. Here, we report that, upon neurodegeneration with okadaic acid, mint-1 and mint-2 levels were reduced by proteolytic cleavage, and that these changes were followed by increases in APP levels. We also show that the mint-1 and mint-2 cleavage and APP overexpression were prevented by calpain inhibitor-I and inhibitor-II. These results indicate that mint cleavage might play a role in the pathophysiology of Alzheimer's disease.

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cadherins; Calpain; Carrier Proteins; Cells, Cultured; Cerebral Cortex; Down-Regulation; Enzyme Inhibitors; Glycoproteins; Membrane Proteins; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Okadaic Acid; Oligopeptides; Rats

Podosomes, highly dynamic adhesion structures implicated in cell motility and extracellular matrix degradation, are characteristic of certain cells of the myeloid lineage and a limited range of other cell types. The nature and the mechanisms that regulate their high turnover are unknown at present. The cysteine protease calpain is involved in the regulation of cell migration in part by promoting either formation or disassembly of adhesion sites. Despite the fact that many known substrates of calpain are also structural components of the podosome complex, no studies have yet demonstrated that calpain participates in the regulation of podosome dynamics. In the present work, we show that inhibition of calpain in primary mouse dendritic cells leads to enhanced accumulation of actin filaments, the Wiskott Aldrich Syndrome protein (WASP), beta(2) integrins, talin, paxillin and vinculin in podosomes. This accumulation of components is associated with stabilisation of podosome turnover, overall reduction in velocity of cell locomotion and impaired transmigration across an endothelial monolayer. We also demonstrate that calpain cleaves the podosome components talin, Pyk2 and WASP in dendritic cells. In summary, our results provide evidence that calpain regulates podosome composition and turnover and that this process is required for efficient migration of dendritic cells.

Topics: Actin Cytoskeleton; Animals; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Line; Cell Membrane Structures; Cell Movement; Cells, Cultured; Dendritic Cells; Enzyme Activation; Focal Adhesion Kinase 2; Glycoproteins; Mice; Mice, Inbred Strains; Oligopeptides; Peptide Fragments; Structure-Activity Relationship; Talin; Wiskott-Aldrich Syndrome Protein

Calpains and endoplasmic reticulum (ER) stress have both been implicated in renal cell death following exposure to reactive chemical toxicants (RCTs). Therefore, we explored the link between ER stress, calpain, and cell death in renal cell injury due to model RCTs (iodoacetamide, menadione, tert-butyl hydroperoxide) and ER stress inducers (tunicamycin [TUN], thapsigargin [THAPS]). The calpain inhibitor, PD150606, significantly reduced the RCT and TUN-induced cell death in the renal cell line LLC-PK1, but not death induced by THAPS. ER stress was confirmed by the significant induction of GRP78 following exposure to RCTs and ER stress inducers. While GRP94 induction was observed following RCTs and TUN, it was not statistically significant because of variability. THAPS at 5 microM significantly induced GRP94, while 20 mmicroM caused a calpain-dependent cleavage of GRP94. Caspase-12 and m-calpain were variably induced and/or cleaved following exposure to all toxicants, supporting activation of these signaling pathways. Inhibition of calpain blocked the induction of GRP78 following exposure to RCTs suggesting that calpain was contributing to the observed ER stress following RCTs. In contrast, calpain inhibition did not block ER stress protein induction following exposure to nontoxic concentrations of TUN or THAPS, indicating that calpain inhibition did not block the ER stress protein induction pathways directly. These studies demonstrate a previously unappreciated link between calpain activation and ER stress-associated cell death in renal cells. While further studies are required to clarify the molecular events involved, these results confirm that calpain activation and the ER are important related players in chemically induced renal cell damage.

Topics: Acrylates; Animals; Calpain; Cell Death; Cell Nucleus; Cell Shape; Cell Survival; Chromatin; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Glycoproteins; Heat-Shock Proteins; Immunoblotting; Iodoacetamide; L-Lactate Dehydrogenase; LLC-PK1 Cells; Membrane Proteins; Oligopeptides; Swine; tert-Butylhydroperoxide; Thapsigargin; Time Factors; Tunicamycin; Vitamin K 3

Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, alpha-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca(2+) concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca(2+) concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5-p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity.

Topics: Animals; Apoptosis; Calcium; Calpain; Carrier Proteins; Cell Survival; Cerebellar Cortex; Cysteine Proteinase Inhibitors; Enzyme Activation; Immunoblotting; Methylmercury Compounds; Microfilament Proteins; Nerve Tissue Proteins; Neurons; Oligopeptides; Rats; Rats, Wistar; Spectrometry, Fluorescence; tau Proteins

At fertilization in mammals, the spermatozoon triggers a unique signal transduction mechanism within the egg, leading to its activation. It is well accepted that the earliest event observed in all activated eggs is an abrupt rise in intracellular calcium concentrations. However, little is known regarding the downstream proteins that are activated by this rise in calcium. Calpains constitute a family of intracellular calcium-dependent cysteine proteases whose members are expressed widely in a variety of cells. We investigated the expression and possible role of the calpain isoforms mu and m throughout egg activation. Both calpains were expressed in the rat egg and localized at the egg cortex as well as in the meiotic spindle. m Calpain translocated to the membrane and to the spindle area during parthenogenetic egg activation and during in vivo fertilization, upon sperm binding to the egg. The cytoskeletal protein alpha-spectrin (fodrin) was proteolysed by calpain during the egg-activation process, as demonstrated by specific calpain-breakdown products. Following parthenogenetic activation by ionomycin or puromycin, the calpain-selective permeable inhibitor, calpeptin, inhibited the resumption of meiosis and cortical reaction in a dose-dependent manner. Calpeptin was also effective in inhibiting in vitro fertilization. These results may imply a correlation between calpain activation and mammalian egg activation at fertilization and a possible role for calpain in the cascade of cellular events leading to resumption of meiosis.

Topics: Animals; Calcium Signaling; Calpain; Dipeptides; Female; Fertilization in Vitro; Fluorescent Antibody Technique; Ionomycin; Ionophores; Male; Meiosis; Microscopy, Confocal; Oligopeptides; Ovum; Parthenogenesis; Puromycin; Rats; Rats, Wistar; Sperm-Ovum Interactions

Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Fusion; Cell Line; Cell Movement; Clone Cells; Cysteine Proteinase Inhibitors; Cytoskeleton; Dipeptides; Dose-Response Relationship, Drug; Glucosidases; Intracellular Signaling Peptides and Proteins; Leupeptins; Membrane Proteins; Mice; Muscle Fibers, Skeletal; Myoblasts; Myristoylated Alanine-Rich C Kinase Substrate; Oligopeptides; Phosphoproteins; Stress Fibers

The ultrastructural change that characterizes the onset of Wallerian degeneration is the disintegration of axoplasmic microtubules and neurofilaments, which are converted into an amorphous and granular material, followed by myelin breakdown. The mechanism underlying such processes is an increase in the amount of intracellular calcium, leading to activation of proteases called calpains. The aim of this study was to evaluate by quantitative ultrastructural analysis whether nerve fibers can be preserved by the use of an exogenous inhibitor of these proteases (calpain inhibitor-2, Mu-F-hF-FMK), after optic nerve crush. For that, the left optic nerves of opossums, Didelphis aurita, were crushed with the aid of a fine forceps, and half of them received a calpain inhibitor mixed with Elvax resin. Ninety-six hours after the lesion, the animals were reanesthetized and transcardially perfused, and the optic nerves were removed, the right ones being used as normal nerves. Afterward, the optic nerves were dissected and processed for routine transmission electron microscopy and quantitative and statistical analysis. The results of this analysis showed that the group that received the calpain inhibitor presented a reduction of astrogliosis, maintaining the optic nerve structure in an organized state; a significant decrease in the number of degenerating fibers; and a significant increase in the number of fibers with preserved cytoskeleton and preservation of axonal and myelin area and integrity, reducing the enlargement and edema of the axon. In conclusion, our findings suggest that calpain inhibitor is able to provide neuroprotection of the central nervous system fibers after a crush lesion.

Topics: Animals; Axons; Calpain; Cysteine Proteinase Inhibitors; Nerve Crush; Nerve Degeneration; Nerve Fibers, Myelinated; Oligopeptides; Opossums; Optic Nerve

Increases in intracellular calcium and subsequent activation of calcium-activated proteases (e.g., calpains) may play a critical role in central nervous system injury. Several studies have implicated calpain activation following subarachnoid hemorrhage (SAH). This study evaluated the effect of a calpain inhibitor administration following SAH in the rat on behavioral deficits (postinjury days 1-5, employing a battery of well-characterized assessment tasks), and blood-brain barrier permeability changes (48 h post-SAH, quantifying the microvascular alterations according to the extravasation of protein-bound Evans Blue using a spectrophotofluorimetric technique). Rats were injected with 400 microl of autologous blood into the cisterna magna to induce SAH. Within 5 min after the surgical procedure, Calpain Inhibitor II or vehicle was continuously administered intravenously for 2 days. Results indicated that Calpain Inhibitor II treatment after SAH significantly improved (a) beam balance time (day 1, p < 0.05), but not beam balance score, (b) latency to traverse the beam on days 1-4 (day 1-3, p < 0.001; day 4, p < 0.01), and (c) loss in body weight on days 4-5 (p < 0.05). Evans Blue dye extravasation was significantly less in SAH Calpain Inhibitor II-treated rats compared to SAH vehicle-treated rats in seven out of the eight brain regions studied (p < 0.001, 0.01, and 0.05). These results suggest that pharmacological inhibition of a relatively selective, membrane-permeant calpain inhibitor can significantly reduce some pathophysiological SAH consequences, and indicate that the inhibition of calpain may be a beneficial therapeutic approach to reduce post-SAH global brain dysfunction.

Topics: Animals; Behavioral Symptoms; Blood-Brain Barrier; Body Weight; Calpain; Male; Motor Skills; Oligopeptides; Permeability; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage

Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action.

Topics: Adipocytes; Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Deoxyglucose; Electric Conductivity; Glucose; In Vitro Techniques; Insulin; Insulin Secretion; Intracellular Membranes; Islets of Langerhans; Leucine; Mice; Mice, Inbred C57BL; Muscle, Skeletal; NADP; Oligopeptides; Osmolar Concentration; Time Factors

N-acetyl-p-benzoquinone imine (NAPQI), a semiquinone metabolite of acetaminophen, produces cataract in mice. Naphthalene is biotransformed to the cataractogenic metabolite 1,2-naphthoquinone (NQ). Intracameral injection of NAPQI elicits a rapid increase in free intracellular Ca2+ in the lens epithelium and calpain activation before lens opacification begins. In order to test whether the cellular response is a common feature of quinone-induced cataracts, we injected in this work 1,2-naphthoquinone (NA) in the anterior chamber of mouse eye and followed cellular responses in the lens prior to opacity development. A marked rise in free intracellular Ca2+ in the lens epithelium and concurrent activation of calpain were observed within 1 hr after NQ injection preceding lens opacity development. These results support the suggestion that Ca2+ release and calpain activation are involved in the mechanism of quinone-induced cataractogenesis.

Topics: Animals; Anterior Chamber; Calcium; Calpain; Cataract; Epithelial Cells; Glycoproteins; Lens, Crystalline; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Naphthoquinones; Oligopeptides

Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, induces growth arrest in a variety of cancer cell lines. Its mechanism of action, however, has not been completely elucidated. E2F-1 is thought to act as an oncogene and a tumour suppressor, with its action probably dependent upon the cellular context. We have shown in this study that transcriptional regulation and proteasomal degradation of E2F-1 are critical regulatory events in lovastatin-induced cell death. Accompanying this is a reduction in the E2F-1-regulated expression of cell cycle genes such as c-myc, cyclin D1, cyclin A and cyclin B1. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease in the S-phase cell population in response to lovastatin. Although expression of E2F-1 was reduced in three prostate cancer cell lines-PC-3, LNCaP and DU-145-the p21 and p27 protein levels were not increased in all the cell lines treated, suggesting that increase in p21 and p27 protein expression per se is not responsible for lovastatin-mediated down-regulation of E2F-1. The subsequent apoptotic death of these cells in the presence of lovastatin can be prevented by forced ectopic expression of E2F-1. Taken together, these facts imply that E2F-1 is the target of an HMG-CoA inhibitor and critical cell death mediator in prostate cancer cells.

Topics: Apoptosis; Calpain; Cell Cycle Proteins; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Male; Oligopeptides; Prostatic Neoplasms; RNA, Messenger; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.

Topics: Acetylcysteine; Acrylates; Amino Acid Chloromethyl Ketones; Apoptosis; Boronic Acids; Bortezomib; Calpain; Cathepsins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Drug Synergism; Genes, p53; Humans; Jurkat Cells; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Neoplasms; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells

The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Base Sequence; Calpain; Caspase 3; Caspase 6; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Etoposide; Humans; Leukemia; Leupeptins; Microtubule-Associated Proteins; Molecular Sequence Data; Mutation; Nucleic Acid Synthesis Inhibitors; Oligopeptides; Protein Serine-Threonine Kinases; Thimerosal; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.

Topics: Actins; Amino Acid Sequence; Animals; Apoptosis; Calpain; Chick Embryo; Ciliary Body; Ciliary Neurotrophic Factor; Cysteine Proteinase Inhibitors; DNA Fragmentation; Ganglia, Parasympathetic; Glycoproteins; Hydrolysis; Molecular Sequence Data; Nerve Tissue Proteins; Oligopeptides; Peptide Fragments; Rats; Rats, Wistar; Sequence Analysis

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/alpha-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca2+-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EGTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.

Topics: Calcimycin; Calpain; Carrier Proteins; Cell Death; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Intermediate Filament Proteins; Leupeptins; Nerve Tissue Proteins; Neuroblastoma; Neurofibrils; Oligopeptides; Tumor Cells, Cultured

Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and beta-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. beta-Tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6-24 h of mitogen depletion (i.e., at least 24-36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.

Topics: Acetylcysteine; Adenosine Triphosphatases; Animals; Calpain; Carrier Proteins; Cell Differentiation; Cell Line; Creatine Kinase; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Iron-Binding Proteins; Multienzyme Complexes; Muscles; Myogenin; Oligopeptides; Proteasome Endopeptidase Complex; Rats; Transferrin-Binding Proteins

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

Topics: Animals; Brain Injuries; Calpain; Cerebral Cortex; Cysteine Proteinase Inhibitors; Functional Laterality; Male; Neurofilament Proteins; Oligopeptides; Rats; Rats, Sprague-Dawley; Spectrin

We have studied specific effects of proteasome inhibition on cell cycle progression. To this end, the protease inhibitors MG115, calpain inhibitor I, and calpain inhibitor II, which display differential inhibitory effects on proteasomes, were used. Cell kinetic studies using bromodeoxyuridine pulse labeling revealed a complete block of G1/S and metaphase transitions and a delayed progression through S phase in cell cultures treated with 54 microM of MG115. Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetics. Calpain inhibitor II and MG2M, which is a structural analogue of MG115, had no effect on the cell cycle. The inhibitory effect of MG115 treatment was reversible, because the cell cycle was immediately resumed when the MG115-containing culture medium was replaced by fresh culture medium. Because ubiquitinated proteins accumulated after MG115 treatment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked. By comparison of biochemical and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics.

Topics: Animals; Bromodeoxyuridine; Calpain; Cell Cycle; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Glycoproteins; Humans; Kinetics; Leupeptins; Microscopy, Phase-Contrast; Multienzyme Complexes; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.

Topics: Adrenergic beta-Agonists; Animals; Calpain; Cell Separation; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cysteine Proteinase Inhibitors; Epithelial Cells; Epithelium; Oligopeptides; Phosphorylation; Pulmonary Alveoli; Rats; Terbutaline

Although several signal transduction pathways, including activation of specific protein kinases have been proposed and studied for the secretory processes of lung surfactant from alveolar epithelial type II cells, the role of proteolytic processing by calpains (calcium-activated neutral proteases) in secretion has not been investigated. Therefore, we examined the effect of cell permeable calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal) and II (N-acetyl-leucyl-leucyl-methioninal) on secretion to test the hypothesis that calpains participate in the secretory processes of alveolar epithelial type II cells. Calpain inhibitor I preferentially inhibits micro (mu)-calpain while inhibitor II inhibits milli (m)-calpain. Isolated type II cells were prelabelled with [3H]-choline for 18-24 h. To measure secretion, [3H]-labelled disaturated phosphatidylcholine (DSPC) released in the medium was monitored. Basal secretion of DSPC was maximally (87%) depressed by the presence of 10 microM inhibitor II. Secretagogue-stimulated secretion was also modulated by inhibitor II treatment. Stimulation with calcium ionophore A23187 enhanced secretion 3-fold. However, cells pre-exposed to inhibitor II displayed a 90% reduction of calcium-stimulated secretion. Terbutaline (10 microM) and ATP (1 mM) each increased secretion 2- and 4-fold, respectively. However, the inhibitor-treated cells, exposed to the same stimuli, attained only 53 or 62% of these increases. Calpain inhibitor I, on the other hand, inhibited neither basal nor stimulated secretion. The results suggest that m-calpain, the major isozyme of lung calpain requiring mM calcium for activity in vitro, is involved in the secretory pathways of alveolar epithelial type II cells.

Topics: Amino Acid Sequence; Animals; Calcium; Calpain; Cell Membrane Permeability; Epithelial Cells; Epithelium; In Vitro Techniques; Leupeptins; Molecular Sequence Data; Oligopeptides; Phosphatidylcholines; Protease Inhibitors; Pulmonary Alveoli; Rats

The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of beta-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC50 values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.

Topics: Amino Acid Sequence; Animals; Calpain; Cattle; Cysteine Endopeptidases; Dipeptides; Kinetics; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Pituitary Gland; Protease Inhibitors; Proteasome Endopeptidase Complex; Substrate Specificity