Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis.

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

An in silico functional annotation and screening of potential drug targets derived from Leishmania spp. hypothetical proteins identified by immunoproteomics.

Leishmaniasis is a parasitic disease caused by the protozoan of the Leishmania genus. While no human vaccine is available, drugs such as pentavalent antimonials, pentamidine and amphotericin B are used for treat the patients. However, the high toxicity of these pharmaceutics, the emergence of parasite resistance and/or their high cost have showed to the urgent need of identify new targets to be employed in the improvement of the treatment against leishmaniasis. In a recent immunoproteomics approach performed in the Leishmania infantum species, 104 antigenic proteins were recognized by antibodies in sera of visceral leishmaniasis (VL) dogs. Some of them were later showed to be effective diagnostic markers and/or vaccine candidates against the disease. Between these proteins, 24 considered as hypothetical were identified in the promastigote and amastigote-like extracts of the parasites. The present study aimed to use bioinformatics tools to select new drug targets between these hypothetical proteins. Their cellular localization was predicted to be seven membrane proteins, as well as eight cytoplasmic, three nuclear, one mitochondrial and five proteins remained unclassified. Their functions were predicted as being two transport proteins, as well as five with metabolic activity, three as cell signaling and fourteen proteins remained unclassified. Ten hypothetical proteins were well-annotated and compared to their homology regarding to human proteins. Two proteins, a calpain-like and clavaminate synthase-like proteins were selected by using Docking analysis as being possible drug targets. In this sense, the present study showed the employ of new strategies to select possible drug candidates, according their localization and biological function in Leishmania parasites, aiming to treat against VL.

Neuron-microglia interaction induced bi-directional cytotoxicity associated with calpain activation.

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

IFN-ß affects the angiogenic potential of circulating angiogenic cells by activating calpain 1.

Circulating angiogenic cells (CACs) are monocyte-derived cells with endothelial characteristics, which contribute to both angiogenesis and arteriogenesis in a paracrine way. Interferon-ß (IFN-ß) is known to inhibit these divergent processes in animals and patients. We hypothesized that IFN-ß might act by affecting the differentiation and function of CACs. CACs were cultured from peripheral blood mononuclear cells and phenotypically characterized by surface expression of monocytic and endothelial markers. IFN-ß significantly reduced the number of CACs by 18-64%. Apoptosis was not induced by IFN-ß, neither in mononuclear cells during differentiation, nor after maturation to CACs. Rather, IFN-ß impaired adhesion to, and spreading on, fibronectin, which was dependent on α5ß1 (VLA-5)-integrin. IFN-ß affected the function of VLA-5 in mature CACs, leading to rounding and detachment of cells, by induction of calpain 1 activity. Cell rounding and detachment was completely reversed by inhibition of calpain 1 activity in mature CACs. During in vitro capillary formation, CAC addition and calpain 1 inhibition enhanced sprouting of endothelial cells to a comparable extent, but were not sufficient to rescue tube formation in the presence of IFN-ß. We show that the IFN-ß-induced reduction of the numbers of in vitro differentiated CACs is based on activation of calpain 1, resulting in an attenuated adhesion to extracellular matrix proteins via VLA-5. In vivo, this could lead to inhibition of vessel formation due to reduction of the locally recruited CAC numbers and their paracrine angiogenic factors.

Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo.

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a ß-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by µ-calpain in vitro. Furthermore, we demonstrated that µ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

Cleavage of IκBα by calpain induces myocardial NF-κB activation, TNF-α expression, and cardiac dysfunction in septic mice.

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.

Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna.

Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.

Chronic high glucose downregulates mitochondrial calpain 10 and contributes to renal cell death and diabetes-induced renal injury.

Whereas most calpains are cytosolic proteases, calpain 10 is resident in mitochondria and is important in mitochondrial homeostasis. Because calpain 10 has been implicated in type 2 diabetes, we studied its possible role in diabetes-induced renal dysfunction. We treated renal proximal tubular cells with high glucose (17 mmol/l) and found decreased mitochondrial calpain 10 mRNA and protein at 96 h compared with cells incubated with 0 or 5 mmol/l glucose or 17 mmol/l D-mannitol. High glucose increased mitochondrial calpain 10 substrates (NDUFB8 and ATP synthase ß), decreased basal and uncoupled respiration, and initiated cell apoptosis as indicated by cleaved caspase 3 and nuclear condensation. Renal calpain 10 protein and mRNA were specifically decreased in streptozotocin-induced diabetic rats with kidney dysfunction, and in diabetic ob/ob mice. In agreement with our in vitro data, the kidneys of streptozotocin-induced diabetic rats had elevated calpain 10 substrates and cleaved caspase 3. Finally, specific siRNA-induced knockdown of calpain 10 in the proximal tubules of control rats resulted in decreased renal function as evidenced by increased serum creatinine, and increased caspase 3 cleavage compared with rats receiving scrambled siRNA. Thus, the glucose-induced loss of calpain 10 in vivo results in renal cell apoptosis and organ failure through accumulation of mitochondrial calpain 10 substrates and mitochondrial dysfunction. Whether this is a major cause of the decreased renal function in diabetic nephropathy will require further studies.

Programmed cellular necrosis mediated by the pore-forming alpha-toxin from Clostridium septicum.

Programmed necrosis is a mechanism of cell death that has been described for neuronal excitotoxicity and ischemia/reperfusion injury, but has not been extensively studied in the context of exposure to bacterial exotoxins. The alpha-toxin of Clostridium septicum is a beta-barrel pore-forming toxin and a potent cytotoxin; however, the mechanism by which it induces cell death has not been elucidated in detail. We report that alpha-toxin formed Ca(2+)-permeable pores in murine myoblast cells, leading to an increase in intracellular Ca(2+) levels. This Ca(2+) influx did not induce apoptosis, as has been described for other small pore-forming toxins, but a cascade of events consistent with programmed necrosis. Ca(2+) influx was associated with calpain activation and release of cathepsins from lysosomes. We also observed deregulation of mitochondrial activity, leading to increased ROS levels, and dramatically reduced levels of ATP. Finally, the immunostimulatory histone binding protein HMGB1 was found to be released from the nuclei of alpha-toxin-treated cells. Collectively, these data show that alpha-toxin initiates a multifaceted necrotic cell death response that is consistent with its essential role in C. septicum-mediated myonecrosis and sepsis. We postulate that cellular intoxication with pore-forming toxins may be a major mechanism by which programmed necrosis is induced.

Neuroprotective effect of Salvia sahendica is mediated by restoration of mitochondrial function and inhibition of endoplasmic reticulum stress.

Herein, we investigated the protective effect of Salvia sahendica against H(2)O(2)-induced cell death in rat pheochromocytoma (PC12) cells. Our data show that S. sahendica blocks apoptosis pathway by inhibition of cytochrome c release from mitochondria and leakage of calcium from endoplasmic reticulum. It also activates/inactivates two members of Bcl-2 family, Bax and Bcl-2. Bax inhibition and Bcl-2 activation suppress release of cytochrome c from mitochondria that prevents cleavage of caspase-3. Besides S. sahendica suppresses ER stress via attenuation of intracellular levels of calcium. Suppression of ER stress decreased calpain activation and subsequently cleavage of caspase-12. Altogether, these results indicate that S. sahendica protects PC12 cells treated with H(2)O(2) via suppression of upstream factors of apoptosis pathway. While oxidative stress is an early event in Alzheimer disease, it seems that S. sahendica prevents deleterious effects of reactive oxygen species by stabilizing mitochondrial membranes and inhibiting ER stress.

RPE-derived exosomes rescue the photoreceptors during retina degeneration: an intraocular approach to deliver exosomes into the subretinal space.

Retinal degeneration (RD) refers to a group of blinding retinopathies leading to the progressive photoreceptor demise and vision loss. Treatments against this debilitating disease are urgently needed. Intraocular delivery of exosomes represents an innovative therapeutic strategy against RD. In this study, we aimed to determine whether the subretinal delivery of RPE-derived exosomes (RPE-Exos) can prevent the photoreceptor death in RD. RD was induced in C57BL6 mice by MNU administration. These MNU administered mice received a single subretinal injection of RPE-Exos. Two weeks later, the RPE-Exos induced effects were evaluated via functional, morphological, and behavior examinations. Subretinal delivery of RPE-Exos efficiently ameliorates the visual function impairments, and alleviated the structural damages in the retina of MNU administered mice. Moreover, RPE-Exos exert beneficial effects on the electrical response of the inner retinal circuits. Treatment with RPE-Exos suppressed the expression levels of inflammatory factors, and mitigated the oxidative damage, indicating that subretinal delivery of RPE-Exos constructed a cytoprotective microenvironment in the retina of MNU administered mice. Our data suggest that RPE-Exos have therapeutic effects against the visual impairments and photoreceptor death. These findings will enrich our knowledge of RPE-Exos, and highlight the discovery of a promising medication for RD.

Vascular endothelial growth factor receptor-2 inhibition promotes cell death and limits endothelial cell proliferation in a neonatal rodent model of stroke.

BACKGROUND AND PURPOSE: Recent studies in neonatal rodent stroke models suggest that recovery is due in part to upregulation of hypoxia-inducible factor-1-a and its downstream target, vascular endothelial growth factor. Vascular endothelial growth factor is upregulated after a hypoxic insult and is involved in neuronal survival, angiogenesis, and neurogenesis during the recovery process. METHODS: We performed a 1.5-hour transient middle cerebral artery occlusion in 10-day-old rats with injury verified by diffusion-weighted MRI during occlusion to determine the effects of vascular endothelial growth factor receptor-2 (VEGFR2) inhibition on injury, apoptosis, and angiogenesis. Two days after reperfusion, the pups received either the VEGFR inhibitor, SU5416 (10 mg/kg per dose) or vehicle (1% dimethyl sulfoxide) for 3 days. RESULTS: VEGFR2 inhibition worsened injury 7 days after injury when compared with the vehicle-treated and injury-alone groups (P<0.01). Furthermore, receptor inhibition was associated with increased VEGFR2 expression 5 days after injury (P<0.05) and increased spectrin cleavage with a shift in favor of the calpain-mediated, caspase-3-independent cleavage (P<0.01). Increased areas of cleaved caspase-3 staining were seen in treated rats at 7 days (P<0.01) There were no differences in gliosis or macrophage recruitment as measured by glial fibrillary acidic protein and Iba-1 expression at this time point. Lastly, VEGFR2 inhibition did not affect the overall vessel surface area but reduced endothelial cell proliferation in injured caudate. CONCLUSIONS: Inhibition of VEGFR2 signaling worsens injury, affects cell death, and reduces endothelial cell proliferation after neonatal stroke. Injury exacerbation may be in part due to a shift of cell fate from apoptosis to necrosis on the continuum spectrum of cell death as well as effects on angiogenesis in the injured brain.

Interleukin-1alpha treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2.

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.

Modulators of calpain activity: inhibitors and activators as potential drugs.

Introduction: Calpains are intracellular Ca2+-dependent cysteine proteases with 15 known members in the enzyme family. They act as regulatory enzymes, their cleavage modifying the function of their substrates. As their substrates have important roles in many physiological processes, adequate function of calpains is mandatory for normal cellular functions. The adverse operation of them is often related to diseases (e.g. neurodegenerative disorders, cancer, type 2 diabetes mellitus, or limb-girdle muscular dystrophy type 2A).Areas covered: Herein, the authors give an overview of calpains, their structure as well as their physiological and pathological functions. The authors further consider the challenges in calpain inhibitor and activator drug discovery and summarize examples of good candidates. New and applicable strategies are also discussed.Expert opinion: Calpain enzymes are attractive targets for inhibitor or activator design and development. The authors believe this area of research is of high potential, although it has many challenges. For one, the selective and targeted inhibition or activation of calpains is needed. Furthermore, uncontrolled calpain activation may cause more serious side effects and so caution is needed when designing novel therapeutics.

Crosstalk between endothelial cell-specific calpain inhibition and the endothelial-mesenchymal transition via the HSP90/Akt signaling pathway.

HYPOTHESIS: The role of non-cardiomyocytes in cardiac remodeling and fibrosis has not been totally understood until now. This study investigated if endothelial cell (EC)-specific calpain participates in myocardial endothelial injury via the endothelial- mesenchymal transition (EndMT) and in cardiac fibroblasts during cell proliferation, thereby contributing to cardiac fibrosis eventually. METHODS: in vitro cultured mouse cardiac ECs were induced with transforming growth factor (TGF)-ß1 (10 ng/ml) and calpain inhibitor III (20 µM) or Akt inhibitor (LY294002, 20 µM). Isolated cardiac fibroblasts were induced by TGF-ß1 and an HSP90 inhibitor (17AAG, 20 µM), and EndMT were analysed. Capn4-knockout (KO) specific to ECs of mice was generated. We induced the pathological process mimicking cardiac hypertrophy and fibrosis in both Capn4-KO mice and their wild-type littermates. The histological analysis was used to measure cardiomyocyte size and collagen contained in the heart. The immunofluorescence analysis was performed to demonstrate that the ECs went through the EndMT, transforming mesenchymal cells into fibroblasts and myofibroblasts. RESULTS: Capn4 deletion specific to ECs abrogated activity of both calpain 1 and calpain 2 in ECs, lowered the volume of cardiac collagen and cardiomyocytes size, and ameliorated myocardial dysfunction in the isoproterenol-treated cardiac fibrosis model. An ex vivo analysis of cardiomyocytes by Evans Blue staining revealed that isoproterenol increased cell death compared with the control, and Capn4-KO alleviated this result. Inhibiting calpain in cultured cardiac microvascular endothelial cells (MCECs) reversed the EndMT process, which was induced by TGF-ß1. Overexpression of calpastatin decreased the pathological EndMT process, showing that the cultured MCECs have more mesenchymal markers, such as α-smooth muscle actin (SMA), and fewer endothelial markers, such as VE-cadherin. Activating calpain elevated phosphorylated Akt in mice cultured ECs, and inhibiting calpain decreased phosphorylated Akt. Upregulation of phosphorylated Akt by calpain promoted the EndMT, whereas inhibiting calpain switched on the protective mechanism during the EndMT via the heat shock protein (HSP)90/Akt signaling way in cultured ECs. CONCLUSIONS: This study demonstrated a vital role of calpain in ECs for inducing myocardiocyte hypertrophy, cell death and the EndMT via the HSP90/Akt signaling pathway, thereby promoting cardiac fibrosis. The results indicate that inhibiting ECs calpain is a novel therapeutic target to retard cardiac fibrosis and has positive effects on heart failure.

mu-Calpain mediated cleavage of the Na+/Ca2+ exchanger in isolated mitochondria under A23187 induced Ca2+ stimulation.

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.

Alterations in neurofilaments content and calpains activity of sciatic nerve of carbon disulfide-treated rats.

Chronic exposure to carbon disulfide (CS2) can induce polyneuropathy in occupational worker and experimental animals, but underlying mechanism for CS2 neurotoxicity is currently unknown. In the present study, male Wistar rats were randomly divided into two experimental groups and one control group. The rats in two experimental groups were treated with CS2 by gavage at dosages of 300 and 500 mg/kg per day, respectively, five times per week for 12 weeks. The contents of neurofilament triplet proteins (NF-H, NF-M, NF-L) and two calpain isoforms (m-calpain and u-calpain) in sciatic nerves were determined by immunoblotting. In the meantime, the mRNA levels of NF-H, NF-M and NF-L in spinal cords were quantified by reverse transcriptase-polymerase chain reaction, and the total activity of calpains in sciatic nerves was measured by fluorescence assay. Results showed that the contents of NF-M and NF-L in CS2-treated rats sciatic nerves increased significantly except NF-M in low dose group. The contents and activity of m-calpain and u-calpain in sciatic nerve also demonstrated a significant elevation. Furthermore, the levels of mRNA expression of NFH, NFM and NFL genes were up-regulated consistently in spinal cords of treated rats. These findings suggested that CS2 intoxication was associated with the disruption of neurofilaments homeostasis and activiation of calpains in rat sciatic nerves, which might be involved in the development of CS2-induced peripheral neuropathy.

Nimodipine attenuates the parkinsonian neurotoxin, MPTP-induced changes in the calcium binding proteins, calpain and calbindin.

We have recently demonstrated neuroprotective abilities of nimodipine, an L-type voltage dependent calcium channel (VDCC) blocker in cellular and animal models of Parkinson's disease (PD). To understand the calcium regulatory mechanisms in the disease pathogenesis, the present study examined calcium regulatory proteins calbindin and calpain mRNA and protein levels employing quantitative PCR and western blot in 1-methyl-4-phenyl pyridinium ion (MPP+)-treated SH-SY5Y cell lines and in the striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). mRNA and protein levels of calbindin were lower, while that of calpain were higher in MPP+-treated SH-SY5Y cells and MPTP-treated mouse striatum as compared to their respective controls. Nimodipine pretreatment significantly attenuated these effects in the parkinsonian neurotoxin-treated SH-SY5Y cell line and in the mouse striatum. The activities of the apoptotic mediator, caspase-3 and calpain were increased in the neurotoxin-treated groups as compared to their respective controls, which was ameliorated by nimodipine pretreatment. These results suggest that parkinsonian neurotoxin-mediated dopaminergic neuronal death might involve defects in calcium regulatory proteins that control intracellular calcium homeostasis, and these could be corrected by inhibiting L-type VDCC activity. These findings support the notion that hypertensive patients who are on long-term intake of dihydropyridine have reduced risk for PD.

Ingestion of soy protein isolate attenuates eccentric contraction-induced force depression and muscle proteolysis via inhibition of calpain-1 activation in rat fast-twitch skeletal muscle.

OBJECTIVE: Eccentric contraction (ECC) is a contraction in which skeletal muscles are stretched while contracting. The aim of this study was to determine how ingestion of soy protein isolate (SPI) or animal-based proteins affect force deficit, calpain activation, and proteolysis of calcium ion (Ca2+)-regulatory proteins in rat fast-twitch muscles subjected to ECC. METHODS: In the first experiment, male Wistar rats were randomly assigned to a control and an SPI group, which were fed a 20% casein and a 20% SPI diet, respectively, for 28 d before the ECC protocol. Anterior crural muscles underwent 200 repeated ECCs and were excised 3 d later. In the second experiment, half of the SPI rats were given water containing NG-nitro-l-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase, for 3 d of recovery after ECC. RESULTS: SPI ingestion attenuated ECC-induced force deficit, proteolysis of Ca2+-regulatory proteins, and autolysis of calpain-1. Co-ingestion of L-NAME inhibited SPI-associated increases in nitrite and nitrate levels and negated the force recovery effects of SPI. CONCLUSION: These results suggest that SPI ingestion inhibits ECC-elicited force deficit and proteolysis of Ca2+ regulatory proteins, which is caused by inhibited activation of calpain-1 via increased nitric oxide production.