calpain-inhibitor-iii and calpastatin

Recent studies in septic models have shown that myocardial calpain activity and TNF-α expression increase during sepsis and that inhibition of calpain activation downregulates myocardial TNF-α expression and improves cardiac dysfunction. However, the mechanism underlying this pathological process is unclear. Thus, in the present study, we aimed to explore whether IκBα/NF-κB signaling linked myocardial calpain activity and TNF-α expression in septic mice. Adult male mice were injected with LPS (4 mg/kg ip) to induce sepsis. Myocardial calpain activity, IκBα/NF-κB signaling activity, and TNF-α expression were assessed, and myocardial function was evaluated using the Langendorff system. In septic mice, myocardial calpain activity and TNF-α expression were increased and IκBα protein was degraded. Furthermore, NF-κB was activated, as indicated by increased NF-κB p65 phosphorylation, cleavage of p105 into p50, and its nuclear translocation. Administration of the calpain inhibitors calpain inhibitor Ш and PD-150606 prevented the LPS-induced degradation of myocardial IκBα, NF-κB activation, and TNF-α expression and ultimately improved myocardial function. In calpastatin transgenic mice, an endogenous calpain inhibitor and cultured neonatal mouse cardiomyocytes overexpressing calpastatin also inhibited calpain activity, IκBα protein degradation, and NF-κB activation after LPS treatment. In conclusion, myocardial calpain activity was increased in septic mice. Calpain induced myocardial NF-κB activation, TNF-α expression, and myocardial dysfunction in septic mice through IκBα protein cleavage.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Dipeptides; Disease Models, Animal; Heart; Heart Diseases; I-kappa B Proteins; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Myocardium; NF-kappa B; NF-KappaB Inhibitor alpha; Sepsis; Signal Transduction; Tumor Necrosis Factor-alpha

Calpain has been considered to be the most important protease involved in tenderization during the conversion of muscle into meat. However, recent evidence suggests the possible involvement of the key apoptosis protease, caspase, on post-mortem tenderization. This study used inhibitors of calpain and caspase-3 to treat chicken muscle immediately after slaughter and followed the changes in caspase-3 and calpain activities together with their expression during 5 days of aging. Addition of calpain inhibitors to the system resulted in significantly higher caspase-3 activities (p < 0.01) during storage. Western blot analysis of pro-caspase-3 and α-spectrin cleavage of the 120 kDa peptide (SBDP 120) showed that the addition of calpain inhibitors resulted in the formation of higher amounts of the active form of caspase-3 compared with the control (p < 0.01). Inclusion of inhibitors of caspase-3 led to lower calpain activities (p < 0.01) and dramatically reduced the expression of calpain-1 and calpain-2 (p < 0.01). Concomitantly, this inhibition resulted in greater calpastatin expression compared with the control (p < 0.01). The findings of this investigation show that calpain prevented the activation of caspase-3, whereas caspase-3 appeared to enhance the calpain activity during post-mortem aging through inhibition of calpastatin. It is therefore suggested that there is a relationship between caspase-3 and calpain which contributes to the tenderizing process during the conversion of muscle tissue into meat.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Chickens; Dipeptides; Muscle, Skeletal; Oligopeptides; Poultry; Protease Inhibitors

We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na(+)/Ca(2+) exchanger (NCX) in the ER. Treatment of the ER with Ca(2+) (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 microM) or MDL28170 (10 microM), or Ca(2+) chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca(2+) overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Dipeptides; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Immunoprecipitation; In Vitro Techniques; Muscle, Smooth; Protein Binding; Pulmonary Artery; Sodium-Calcium Exchanger

Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 microM) stimulates mu-calpain activity and subsequently cleaves Na(+)/Ca(2+) exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca(2+) chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca(2+) level in the mitochondria, which subsequently dissociates mu-calpain-calpastatin association leading to the activation of mu-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110kDa) resulting in the doublet of approximately 54-56kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified mu-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca(2+) overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle.

Topics: Animals; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Dipeptides; Intracellular Membranes; Mitochondria, Muscle; Mitochondrial Membranes; Muscle, Smooth, Vascular; Phosphatidylcholines; Phospholipid Ethers; Pulmonary Artery; Sodium-Calcium Exchanger

Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-alpha) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-alpha expression, and myocardial dysfunction during LPS stimulation.. In cultured adult rat cardiomyocytes, LPS (1 microg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-alpha expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91(phox)-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-alpha expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-alpha expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively.. gp91(phox)-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-alpha expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Endotoxemia; Heart; Lipopolysaccharides; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Myocytes, Cardiac; NADPH Oxidase 2; NADPH Oxidases; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Tumor Necrosis Factor-alpha

Caspase-independent cell death, an important death pathway in many cells including neurons, is executed via apoptosis-inducing factor (AIF), an oxidoreductase, localized to the mitochondrial intermembrane space. AIF is processed and released from mitochondria following mitochondrial permeability transition pore (mPTP) formation, and translocates to the nucleus to induce DNA fragmentation and cell death. The release of AIF requires cleavage of its N-terminus anchored in the inner mitochondrial membrane. The protease responsible for this AIF truncation has not been established, although there is considerable evidence suggesting a role for micro-calpain. We previously found that a pool of micro-calpain is localized to the mitochondrial intermembrane space, the submitochondrial compartment in which AIF truncation occurs. The close submitochondrial proximity of mitochondrial micro-calpain and AIF gives support to the hypothesis that mitochondrial micro-calpain may be the protease responsible for processing AIF prior to its release. In the present study, AIF was released from rat liver mitochondria following mPTP induction by atractyloside. This release was inhibited by the cysteine protease inhibitor MDL28170, but not by more specific calpain inhibitors PD150606 and calpastatin. Atractyloside caused swelling in rat brain mitochondria, but did not induce AIF release. In a mitochondrial fraction from SH-SY5Y neuroblastoma cells, incubation with 5 mM Ca(2+) resulted in the activation of micro-calpain but not in AIF truncation. In summary, the localization of micro-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive.

Topics: Acrylates; Animals; Apoptosis Inducing Factor; Atractyloside; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cerebral Cortex; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Inhibitors; Liver; Male; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Rats; Rats, Sprague-Dawley

Right ventricular contractile failure from acute RV pressure overload is an important cause of morbidity and mortality, but the mechanism of RV failure in this setting is incompletely defined. We hypothesized that RV dysfunction from acute RV pressure overload is, in part, due to activation of calpain, and that calpain inhibition would therefore attenuate RV dysfunction. Anesthetized, open chest pigs were treated with the calpain inhibitor MDL-28170 or with inactive vehicle, and then subjected to acute RV pressure overload for 90 min. RV contractile function was assessed by the regional Frank-Starling relation. RV myocardial tissue was analyzed for evidence of calpain activation and calpain-mediated proteolysis. RV pressure overload caused severe contractile dysfunction, along with significant alterations in the endogenous calpain inhibitor calpastatin typical of calpain activation. MDL-28170 attenuated RV free wall dysfunction by more than 50%. However, there were no differences in degradation of spectrin, desmin, troponin-I or SERCA2 between SHAM operated pigs and pigs subjected to acute RV pressure overload, or between vehicle and MDL-28170 treated pigs. Acute RV pressure overload causes calpain activation, and RV contractile dysfunction from acute RV pressure overload is attenuated by the calpain inhibitor MDL-28170; however, the effect is not explained by inhibition of calpain-mediated degradation of spectrin, desmin, troponin-I or SERCA2. Because this is the first report of any agent that can directly attenuate RV contractile dysfunction in acute RV pressure overload, further investigation of the mechanism of action of MDL-28170 in this setting is warranted.

Topics: Animals; Calcium-Binding Proteins; Calpain; Densitometry; Desmin; Dipeptides; Female; Hemodynamics; Male; Myocardial Contraction; Protein Processing, Post-Translational; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrin; Swine; Troponin; Ventricular Dysfunction, Right; Ventricular Pressure

This study is aimed at demonstrating the role played by a calpastatin isoform (Xcalp3) in Xenopus embryos. A specific monoclonal antibody (mAb) was raised against a glutathione S-transferase (GST)-Xcalp3 fusion protein and characterized by immunoblotting and confocal fluorescence microscopy on stage 20-36 embryos. Under these conditions, calpastatin reactivity is associated with a major 110kDa protein fraction and preferentially expressed by notochord and somitic cells. In notochord cells, anti-calpastatin reactive sites were initially restricted to the luminal space of the vacuoles and later became diffused throughout the cytoplasm. In contrast, anti-calpastatin reactive sites in somitic cells were initially diffused throughout the cytoplasm and became restricted to a few intracellular granules in the later developmental stages. At the ultrastructural level, notochord cells appeared as flattened discs containing several vacuoles and numerous electron-dense granules. During transition from stages 26 to 32, electron-dense granules were gradually reduced in number as vacuoles enlarged in size and losed their calpastatin reactivity. Electron-dense granules were also present in myoblast cells and their number gradually reduced during development. To determine whether these observations bear any causal relationship to the calpain/calpastatin system, a number of Xenopus embryos were examined both ultrastructurally and histochemically following exposure to a specific calpain inhibitor (CI3). Under these conditions, Xenopus embryos exhibited an altered right-left symmetry and an abnormal axial shortening. In CI3-treated stage 32 embryos, notochord cells had a reduced vacuolar extension and exhibited at the same time an increase in granular content. The overall morphology of the somites was also distorted and myoblasts were altered both in shape and granular content. Based on these findings, it is concluded that the calpain/calpastatin may play an important role in the control of notochord elongation and somite differentiation during Xenopus embryogenesis.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cytoplasm; Dipeptides; Embryo, Nonmammalian; Enzyme Inhibitors; Immunoblotting; Immunohistochemistry; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Myoblasts; Notochord; Protein Isoforms; Somites; Vacuoles; Xenopus laevis

The molecular mechanisms mediating degeneration of midbrain dopamine neurons in Parkinson's disease (PD) are poorly understood. Here, we provide evidence to support a role for the involvement of the calcium-dependent proteases, calpains, in the loss of dopamine neurons in a mouse model of PD. We show that administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) evokes an increase in calpain-mediated proteolysis in nigral dopamine neurons in vivo. Inhibition of calpain proteolysis using either a calpain inhibitor (MDL-28170) or adenovirus-mediated overexpression of the endogenous calpain inhibitor protein, calpastatin, significantly attenuated MPTP-induced loss of nigral dopamine neurons. Commensurate with this neuroprotection, MPTP-induced locomotor deficits were abolished, and markers of striatal postsynaptic activity were normalized in calpain inhibitor-treated mice. However, behavioral improvements in MPTP-treated, calpain inhibited mice did not correlate with restored levels of striatal dopamine. These results suggest that protection against nigral neuron degeneration in PD may be sufficient to facilitate normalized locomotor activity without necessitating striatal reinnervation. Immunohistochemical analyses of postmortem midbrain tissues from human PD cases also displayed evidence of increased calpain-related proteolytic activity that was not evident in age-matched control subjects. Taken together, our findings provide a potentially novel correlation between calpain proteolytic activity in an MPTP model of PD and the etiology of neuronal loss in PD in humans.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adenoviridae; Aged; Aged, 80 and over; Animals; Behavior, Animal; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Excitatory Postsynaptic Potentials; Genetic Vectors; Humans; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Middle Aged; Parkinson Disease; Proto-Oncogene Proteins c-fos; Radioimmunoassay; Striatonigral Degeneration; Substantia Nigra; Tyrosine 3-Monooxygenase

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

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