calpain and calpeptin

The neuroprotective capacity of morin hydrate (MH), a potent antioxidant flavonoid, and calpeptin (CP), a calpain inhibitor, was documented against different insults but not Huntington's disease (HD). Accordingly, we aim to assess the neuroprotective potential of MH and/or CP in a 3-nitropropionic acid (3-NP)-induced HD model. The 3-NP-treated rats were post-treated with saline, MH, CP, or MH + CP for a week. Post-treatment with MH and/or CP amended motor function (beam walking test) and short-/ long-term spatial memory (novel object recognition test) and improved cortical microscopic architecture. On the molecular level, MH, and to a lesser extent CP, inhibited the cortical content/expression of glutamate, calpain, and Kidins220 and abated the inflammatory molecules, nuclear factor (NF)-κB, tumor necrosis factor-α, and interleukin-1β, as well as lipid peroxidation. However, MH, but barely CP, activated the molecules of the neuroprotective trajectory; viz., brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase receptor B (TrkB), protein kinase B (AKT), and cAMP response element-binding protein (CREB). Compared to the single treatments, the combination regimen mediated further reductions in the cortical contents of glutamate, calpain, and Kidins220, effects that extended to entail the anti-inflammatory/anti-oxidant potentials of MH and to a greater extent CP. However, the combination of MH strengthened the fair effect of CP on the survival signaling pathway BDNF/TrkB/AKT/CREB. In conclusion, MH, CP, and especially their combination, afforded neuroprotection against HD through curbing the glutamate/calpain axis, Kidins220, as well as NF-κB-mediated neuroinflammation/oxidative stress, besides activating the BDNF/TrkB/AKT/CREB hub that was partly dependent on calpain inhibition.

Topics: Animals; Antioxidants; Brain-Derived Neurotrophic Factor; Calpain; Cyclic AMP Response Element-Binding Protein; Flavonoids; Glutamic Acid; Membrane Proteins; Neuroprotective Agents; Phosphoproteins; Proto-Oncogene Proteins c-akt; Rats

Diabetes mellitus (DM) has been considered an accelerator of Alzheimer's disease (AD), but the cellular and molecular mechanisms underlying this effect are not fully understood. Here, we attempted to determine the role and regulatory mechanism of calpain in the AD-like cognitive decline and pathological changes in rats caused by DM. In the initial stages, our results verified that DM model rats showed cognitive impairment, as well as a loss of neurons, decreased pericyte marker (PDGFR-β and α-SMA), and calpain-2 expression and amyloid-β (Aβ) deposition in the hippocampal tissues. In high glucose-induced primary pericytes, the cell apoptotic rate was increased, and cell proliferation was inhibited in a time-dependent manner. The protein level of calpain-2 was also upregulated by HG induction, but the level of calpain-1 did not change with HG treatment, which was also observed in DM model rats. Subsequently, some DM model rats were administered calpeptin, an inhibitor of calpain. Our data revealed that calpeptin treatment significantly suppressed calpain-1 and calpain-2 expression in the hippocampal tissues and effectively improved the cognitive impairments of DM model rats. Neuronal loss, Aβ accumulation, pericyte loss, inflammation, and oxidative stress injury in the hippocampal tissues of DM model rats were also partly rescued by calpeptin administration. Our work demonstrated that the calpain inhibitor calpeptin could alleviate DM-induced AD-like cognitive impairments and pathological changes in rats, and this effect may be associated with pericytes. Calpeptin may become a promising drug to treat the AD-like complications of DM.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Calpain; Cognitive Dysfunction; Diabetes Mellitus; Dipeptides; Glucose; Glycoproteins; Rats

This perspective considers the benefits of the potential future use of the cell permeant calpain inhibitor, calpeptin, as a drug to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Recent work has reported calpeptin's capacity to inhibit entry of the virus into cells. Elsewhere, several drugs, including calpeptin, were found to be able to inhibit extracellular vesicle (EV) biogenesis. Unsurprisingly, because of similarities between viral and EV release mechanisms, calpeptin has also been shown to inhibit viral egress. This approach, identifying calpeptin, through large-scale screening studies as a candidate drug to treat COVID-19, however, has not considered the longer term likely benefits of calpain inhibition, post-COVID-19. This perspective will reflect on the capacity of calpeptin for treating long COVID by inhibiting the overproduction of neutrophil extracellular traps potentially damaging lung cells and promoting clotting, together with limiting associated chronic inflammation, tissue damage and pulmonary fibrosis. It will also reflect on the tolerated and detrimental in vivo side-effects of calpain inhibition from various preclinical studies.

Topics: Calpain; COVID-19 Drug Treatment; Humans; Post-Acute COVID-19 Syndrome; SARS-CoV-2

The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60-62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca

Topics: Animals; Apoptosis; Brain; Calcium; Calcium Signaling; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Isoenzymes; Leupeptins; Male; Mitochondria; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Molecular Weight; Muscle Proteins; Phosphorylation; Protein Transport; Rats

Filamin A is the most widely expressed isoform of filamin in mammalian tissues. It can be hydrolyzed by Calpain, producing a 90-kDa carboxyl-terminal fragment (ABP90). Calpeptin is a chemical inhibitor of Calpain, which can inhibit this effect. It has been shown that ABP90 acts as a transcription factor which is involved in mediating cell signaling. However, the significance of ABP90 and its clinical signature with underlying mechanisms have not been well studied in glioblastoma multiforme (GBM).. ABP90 protein was measured in 36 glioma patients by Western blot. Human GBM cell lines U87 and A172 were used to clarify the precise role of ABP90. CCK-8 assay was used to analyze the cell viability. Transwell invasion assay and wound healing assay were used to analyze the migration and invasion. Expression of matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot.. ABP90 protein expression was lower in GBM tissues. The patients with low ABP90 protein expression had a shorter OS time (p = 0.046). After being treated with Calpain, the expression of ABP90 was upregulated, which led to a decline of cell viability, enhanced the efficacy of temozolomide and restrained the cell invasion. Calpeptin could inhibit the effect. The mechanism might be involved in the balance of MMP2/TIMP2.. Our present data suggest that ABP90 expression is a significant prognostic factor and may play an important role in cell viability, chemotherapeutic sensitivity and invasion of GBM.

Topics: Biomarkers, Tumor; Brain Neoplasms; Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dipeptides; Filamins; Glioblastoma; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Prognosis; Temozolomide; Tissue Inhibitor of Metalloproteinase-2

Protease activity correlates with depressive or suicidal behaviors, with calpain activation being especially implicated in depression-like behaviors. However, the role of calpain in depression-like behaviors is currently unknown. In this study, the lipopolysaccharide (LPS) - and chronic unpredictable mild stress (CUMS)-induced depression models were used to evaluate the antidepressant effects of calpain inhibitors. Potential mechanisms were determined using pharmacological and biochemical methods. We found that i. p. injection of a calpain inhibitor, calpeptin, prevented LPS-induced depression-like behaviors, activation of astrocytes, inflammation, and reduction of synaptic protein expression levels. LPS injection (i.p.) promoted calpain activity, which degraded suprachiasmatic nucleus circadian oscillatory protein (SCOP). This led to the activation of ERK and nuclear translocation of nuclear factor kappa-B (NF-κB), the promotion of cytokine release, and the reduction of Arc, and PSD95 expression in the hippocampus. In contrast, i. p. injection of calpeptin blocked these changes. Furthermore, intraventricular injection of calpain inhibitor (PD150606) or an ERK inhibitor ameliorated the LPS-induced depression-like behaviors. Administration of calpeptin also remedied CUMS-induced depression-like behaviors, degradation of SCOP, activation of astrocytes, and reduction of synaptic protein expression levels. Finally, we also demonstrated that memantine, an N-methyl-d-aspartic acid (NMDA) receptor antagonist blocks LPS-induced degradation of SCOP. Together, our results show that calpain inhibition ameliorates depression-like behaviors, probably by reducing inflammation and promoting synaptic protein expression in the hippocampus.

Topics: Animals; Antidepressive Agents; Brain-Derived Neurotrophic Factor; Calpain; Cytoskeletal Proteins; Depression; Dipeptides; Disks Large Homolog 4 Protein; Gene Expression; Glycoproteins; Hippocampus; Inflammation; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Presynaptic Terminals

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Differentiation; Cell Line; Cell Survival; Cells, Cultured; Dipeptides; Disease Models, Animal; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Mutant Strains; Microfilament Proteins; Microtubule-Associated Proteins; Motor Neurons; Muscular Atrophy, Spinal; Proteolysis; Spinal Cord; Survival of Motor Neuron 1 Protein

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

Topics: Algorithms; Calcimycin; Calpain; Cell Line, Tumor; Dipeptides; Humans; MAP Kinase Kinase Kinase 2; Mutagenesis, Site-Directed; Phosphorylation; Protein Serine-Threonine Kinases; Protein Stability; Proteolysis; RNA Interference; RNA, Small Interfering; Temperature

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy.

Topics: Animals; Calpain; Cells, Cultured; Dipeptides; Gene Knockdown Techniques; Glycoproteins; Membrane Potentials; Mice, Transgenic; Motor Activity; Motor Neurons; Muscular Atrophy, Spinal; Mutation; Nerve Degeneration; Neurites; Phenotype; Potassium; Spinal Cord; Survival of Motor Neuron 1 Protein

HIV-1 gp120, an important subunit of the envelope spikes that decorate the surface of virions, is known to play a vital role in neuronal injury during HIV-1-associated neurocognitive disorder (HAND), although the pathological mechanism is not fully understood. Our previous studies have suggested that the V3 loop of HIV-1 gp120 (HIV-1 gp120 V3 loop) can induce neuronal apoptosis in the hippocampus, resulting in impairment in spatial learning and memory in Sprague-Dawley (SD) rats. In this study, we demonstrated that autophagy was significantly increased in rat primary hippocampal neurons in response to treatment of HIV-1 gp120 V3 loop. Importantly, HIV-1 gp120 V3 loop-induced autophagy played a dual role in the cell survival and death. An increase in autophagy for a short period inhibited apoptosis of neurons, while persistent autophagy over an extended period of time played a detrimental role by augmenting the apoptotic cascade in rat primary hippocampal neurons. In addition, we found that the HIV-1 gp120 V3 loop induced autophagy via AMPK/mTOR-dependent and calpain/mTOR-independent pathways, and the ERK/mTOR pathway plays a partial role. These findings provide evidence that HIV-1-induced autophagy plays a dual role in the survival and apoptosis of the primary rat hippocampal neurons and persistent autophagy may contribute to the pathogenesis of HAND, and autophagy modulation may represent a potential therapeutic strategy for reducing neuronal damage in HAND.

Topics: Adenine; Amino Acid Sequence; AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Hippocampus; HIV Envelope Protein gp120; HIV-1; Male; Neurons; Neuroprotective Agents; Peptide Fragments; Protein Kinase Inhibitors; Rats, Sprague-Dawley

Pleural fibrosis is associated with various inflammatory processes such as tuberculous pleurisy and bacterial empyema. There is currently no ideal therapeutic to attenuate pleural fibrosis. Some pro-fibrogenic mediators induce fibrosis through inflammatory processes, suggesting that blockage of these mediators might prevent pleural fibrosis. The MeT-5A human pleural mesothelial cell line (PMC) was used in this study as an in vitro model of fibrosis; and intra-pleural injection of bleomycin with carbon particles was used as an in vivo mouse model of pleural fibrosis. Calpain knockout mice, calpain inhibitor (calpeptin), and angiotensin (Ang) II type 1 receptor (AT

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Bleomycin; Calpain; Carbon; Cell Line; Collagen Type I; Dipeptides; Disease Models, Animal; Fibrosis; Humans; Losartan; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pleural Diseases

The aim of this study is to explore the potent neuroprotective effect of calpeptin (CP) on neuron damage induced by acrylamide (ACR) and its mechanism. Behavioural indicators such as hind limb splay, rota-rod performance, and gait analysis were assessed weekly to evaluate neurobehavioural changes after ACR and/or CP administration. The histopathological alterations and the changes of μ-calpain, m-calpain, microtubule-associated protein 2 (MAP2), and α-tubulin and β-tubulin protein levels in spinal cord were determined. Results showed that after administration of 30 mg/kg ACR, decreased body weight, attenuated neurobehavioural function, injury of motor neuron, increased protein levels of m-calpain and β-tubulin, suppressed MAP2 protein level, and no significant changes of μ-calpain and α-tubulin protein levels were observed compared with the control group rats. After administration of 200 μg/kg CP, partially restored body weight and neurobehavioural function, improvement of motor neuron injury, decreased protein levels of m- calpain and β-tubulin, and reversed effects of MAP2 protein level were observed compared with the ACR group rats. Our results suggested that CP alleviates neuropathy induced by ACR in rats. The calpain's overactivation causes the degrading of MAP2 and eventually leads to the destruction of microtubules (MTs), which may be one of the mechanisms of cytoskeletal damage induced by ACR.

Topics: Acrylamide; Animals; Calpain; Dipeptides; Female; Motor Neuron Disease; Neuroprotective Agents; Rats; Rats, Wistar

Filamin A (FLNA) is the most abundant and widely expressed isoform of filamin in human tissues. It is cleaved by calpain at the hinge 1 and 2 domains, producing a 90-kDa carboxyl-terminal fragment (FLNA. Human melanoma (A7), prostate cancer (PC3), mouse fibrosarcoma (T241) and endothelial (MS1) cells were assayed for proliferation, migration, invasion and colony formation after treatment with calpeptin. Cell lysates were immunoblotted for FLNA and FLNA. These data suggest that the cleavage of FLNA by calpain is an important cellular event in the regulation of tumor cell growth.

Topics: Animals; Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dipeptides; Filamins; Glycoproteins; Humans; Male; Mice; Neoplasms; Proteolysis

Calpains are intracellular calcium-dependent cysteine proteases, which cleave several substrates proteins, have been proven to play important roles in lung fibrosis. The aim of this study was to investigate the effects of calpain on bleomycin (BLM)-induced pulmonary fibrosis. A lung fibrosis mice model was established successfully by intraperitoneal injection of bleomycin. Calpeptin, a highly selective inhibitor of calpain activation, was administered three times weekly after bleomycin injection. Histological examination was used to assess the fibrosis. Quantitative-PCR and Western blotting were used to assess the development of epithelial-mesenchymal transition (EMT). We found calpeptin treatment decreased the BLM-induced EMT-associated markers, such as muscle actin (α-SMA) and collagen-I, while increased E-cadherin (E-cad). Calpeptin also suppressed the activation of transforming growth factor β1 (TGFβ1)-Smad2/3 signaling pathway, which plays crucial role in lung fibrosis and EMT. Furthermore, we found differentiated embryonic chondrocyte-expressed gene 1 (DEC1), an important transcription factor, was upregulated in both patients with idiopathic pulmonary fibrosis and in bleomycin-induced lung fibrosis. DEC1 was suppressed by calpeptin in bleomycin-induced mice model. Collectively, these findings indicated that calpeptin had a potential anti-fibrosis effect, which focus on the development of EMT.

Topics: Actins; Animals; Basic Helix-Loop-Helix Transcription Factors; Bleomycin; Cadherins; Calpain; Collagen Type I; Dipeptides; Epithelial-Mesenchymal Transition; Homeodomain Proteins; Humans; Male; Mice, Inbred C57BL; Pulmonary Fibrosis

The hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive.. We tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes.. Compared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects.. We elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.

Topics: Analysis of Variance; Animals; Blotting, Western; Calcineurin; Calcium; Calpain; Case-Control Studies; Cells, Cultured; Dipeptides; Disease Models, Animal; Gene Expression Regulation; Glomerulosclerosis, Focal Segmental; Humans; Male; Podocytes; Proteinuria; Random Allocation; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reference Values; Signal Transduction; TRPC Cation Channels; TRPC6 Cation Channel; Up-Regulation

Exposure to cigarette smoke including secondhand smoking is the most important risk factor in the development of chronic obstructive pulmonary disease where incidence has substantially increased in recent decades. The mechanisms responsible for cigarette smoke-induced pulmonary inflammation remain unclear, and thus lack of effective treatment. The present study investigated the effect of calpeptin on attenuating cigarette smoke induced pulmonary inflammation and its potential mechanism and function. When BALB/c mice were exposed to cigarette smoke and received calpeptin intraperitoneally injection after 90 days, calpeptin histologically attenuated the accumulation of neutrophils (P < 0.001), eosinophils (P < 0.001), macrophages (P < 0.01), fibrinous exudation and proliferation within the interstitial and alveolar spaces. BEAS-2B cells were added with cigarette smoke extract in vitro and treated with calpeptin for 24 h in the treatment group. The markedly upregulation of μ-calpain (P < 0.01), m-calpain (P < 0.001) and IκBα (P < 0.01) in cigarette smoke-induced lungs were simultaneously decreased by calpeptin treatment (P < 0.05). The increased expression of μ-calpain, m-calpain and IκBα (P < 0.05) in cigarette smoke extract-stimulated BEAS-2B cells were also decreased by calpeptin treatment (P < 0.05). These data indicated that calpeptin attenuated cigarette smoke-induced pulmonary inflammation by suppressing the pathway of μ-calpain, m-calpain and IκBα in vivo and in vitro. Calpeptin might have a potential for prevention of the development of inflammatory pulmonary diseases and warrant further pharmaceutical investigation.

Topics: Animals; Calpain; Cell Line; Dipeptides; Humans; Male; Mice; Mice, Inbred BALB C; NF-KappaB Inhibitor alpha; Pneumonia; Signal Transduction; Tobacco Smoke Pollution

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.

Topics: Animals; Calpain; Cytoskeleton; Dipeptides; Encephalomyelitis, Autoimmune, Experimental; Injections, Spinal; Male; Neurons; Oligopeptides; Proteasome Endopeptidase Complex; Protein Carbonylation; Proteolysis; Rats; Rats, Inbred Lew

Chronic rotator cuff (RC) tears are characterized by retraction, fat accumulation, and atrophy of the affected muscle. These features pose an intractable problem for surgical repair and subsequent recovery, and their prevention may be easier than reversal. Using an established ovine model, we tested the hypothesis that inhibition of the protease calpain mitigates m. infraspinatus atrophy by preservation of the myofibers' structural anchors in the sarcolemma (the costameres). Already 2 weeks of distal tendon release led to a reduction in muscle volume (-11.6 ± 9.1 cm

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Female; Muscular Atrophy; Rotator Cuff Injuries; Sarcolemma; Sheep

Methylmercury (MeHg) is an environmental pollutant that shows severe toxicity to humans and animals. However, the molecular mechanisms mediating MeHg toxicity are not completely understood. We have previously reported that the MARCKS protein is involved in the MeHg toxicity to SH-SY5Y neuroblastoma and EA.hy926 vascular endothelial cell lines. In addition, calpain, a Ca

Topics: Calcium; Calpain; Cell Line; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Dipeptides; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Methylmercury Compounds; Myristoylated Alanine-Rich C Kinase Substrate; Proteolysis; Signal Transduction

Allergic asthma is characterized by inflammation and airway remodelling. Airway remodelling with excessive deposition of extracellular matrix (ECM) and larger smooth muscle mass are correlated with increased airway responsiveness and asthma severity. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodelling. However, the role of calpain in airway smooth muscle remodelling remains unknown.. To investigate the role of calpain in asthmatic airway remodelling as well as the underlying mechanism.. The mouse asthma model was made by ovalbumin sensitization and challenge. Calpain conditional knockout mice were studied in the model. Airway smooth muscle cells (ASMCs) were isolated from smooth muscle bundles in airway of rats. Cytokines IL-4, IL-5, TNF-α, and TGF-β1, and serum from patients with asthma were selected to treated ASMCs. Collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs were analysed.. Inhibition of calpain using calpain knockout mice attenuated airway smooth muscle remodelling in mouse asthma models. Cytokines IL-4, IL-5, TNF-α, and TGF-β1, and serum from patients with asthma increased collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs, which were blocked by the calpain inhibitor MDL28170. Moreover, MDL28170 reduced cytokine-induced increases in Rictor protein, which is the most important component of mammalian target of rapamycin complex 2 (mTORC2). Blockage of the mTORC2 signal pathway prevented cytokine-induced phosphorylation of Akt, collagen-I synthesis, and cell proliferation of ASMCs and attenuated airway smooth muscle remodelling in mouse asthma models.. Our results indicate that calpain mediates cytokine-induced collagen-I synthesis and proliferation of ASMCs via the mTORC2/Akt signalling pathway, thereby regulating airway smooth muscle remodelling in asthma.

Topics: Airway Remodeling; Animals; Asthma; Calpain; Cell Proliferation; Collagen Type I; Cytokines; Dipeptides; Disease Models, Animal; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Knockout; Muscle, Smooth; Myocytes, Smooth Muscle; Phosphorylation; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Signal Transduction

In addition to protein synthesis, protein degradation or protein cleavage may be necessary for intermediate (ITM) and long-term memory (LTM) to remove molecular constraints, facilitate persistent kinase activity and modulate synaptic plasticity. Calpains, a family of conserved calcium dependent cysteine proteases, modulate synaptic function through protein cleavage. We used the marine mollusk Aplysia californica to investigate the in vivo role of calpains during intermediate and long-term operant memory formation using the learning that food is inedible (LFI) paradigm. A single LFI training session, in which the animal associates a specific netted seaweed with the failure to swallow, generates short (30min), intermediate (4-6h) and long-term (24h) memory. Using the calpain inhibitors calpeptin and MDL-28170, we found that ITM requires calpain activity for induction and consolidation similar to the previously reported requirements for persistent protein kinase C activity in intermediate-term LFI memory. The induction of LTM also required calpain activity. In contrast to ITM, calpain activity was not necessary for the molecular consolidation of LTM. Surprisingly, six hours after LFI training we found that calpain activity was necessary for LTM, although this is a time at which neither persistent PKC activity nor protein synthesis is required for the maintenance of long-term LFI memory. These results demonstrate that calpains function in multiple roles in vivo during associative memory formation.

Topics: Animals; Aplysia; Association Learning; Calpain; Conditioning, Operant; Cysteine Proteinase Inhibitors; Dipeptides; Memory

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

Topics: Adult; Biomarkers; Blood Glucose; Blood Platelets; Calpain; Case-Control Studies; Circulating MicroRNA; Cross-Sectional Studies; Cysteine Proteinase Inhibitors; DEAD-box RNA Helicases; Diabetes Mellitus, Type 2; Dipeptides; Down-Regulation; Female; Humans; K562 Cells; Male; Megakaryocytes; Middle Aged; P-Selectin; Platelet Activation; Receptors, Purinergic P2Y12; Ribonuclease III; Time Factors; Transfection; Up-Regulation

The permeability transition pore (PTP) of inner mitochondrial membranes is a large conductance pathway for ions up to 1500 Da which opening is responsible for ion equilibration and loss of membrane potential in apoptosis and thus in several neurodegenerative diseases. The PTP can be regulated by the Ca(2+)-activated mitochondrial K channel (BK). Calpains are Ca(2+)-activated cystein proteases; calpeptin is an inhibitor of calpains. We wondered whether calpain or calpeptin can modulate activity of PTP or BK. Patch clamp experiments were performed on mitoplasts of rat liver (PTP) and of an astrocytoma cell line (BK). Channel-independent open probability (P(o)) was determined (PTP) and, taking into account the number of open levels, NP(o) by single channel analysis (BK). We find that PTP in the presence of Ca(2+) (200 μM) is uninfluenced by calpain (13 nM) and shows insignificant decrease by the calpain inhibitor calpeptin (1 μM). The NP(o) of the BK is insensitive to calpain (54 nM), too. However, it is significantly and reversibly inhibited by the calpain inhibitor calpeptin (IC50 = 42 μM). The results agree with calpeptin-induced activation of the PTP via inhibition of the BK. Screening experiments with respirometry show calpeptin effects, fitting to inhibition of the BK by calpeptin, and strong inhibition of state 3 respiration.

Topics: Animals; Astrocytes; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Humans; Large-Conductance Calcium-Activated Potassium Channels; Mice, Inbred C57BL; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Patch-Clamp Techniques; Rats, Wistar

Desmoplasia contributes to the aggressive behavior of pancreatic cancer. However, recent clinical trials testing several antifibrotic agents on pancreatic cancer have not shown clear efficacy. Therefore, further investigation of desmoplasia-targeting antifibrotic agents by another mechanism is needed. Calpeptin, an inhibitor of calpains, suppressed fibroblast function and inhibited fibrosis. In this study, we investigated the anticancer effects of calpeptin on pancreatic cancer. We investigated whether calpeptin inhibited tumor progression using a mouse xenograft model. We used quantitative RT-PCR to evaluate the expression of calpain-1 and calpain-2 mRNA in pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). We also undertook functional assays, including proliferation, migration, and invasion, to evaluate the inhibitory effects of calpeptin on PCCs and PSCs. Quantitative RT-PCR indicated that PCCs and PSCs expressed calpain-2 mRNA. Calpeptin reduced tumor volume (P = 0.0473) and tumor weight (P = 0.0471) and inhibited the tumor desmoplastic reaction (P < 0.001) in xenograft tumors in nude mice. Calpeptin also inhibited the biologic functions of PCCs and PSCs including proliferation (P = 0.017), migration (P = 0.027), and invasion (P = 0.035) in vitro. Furthermore, calpeptin reduced the migration of PCCs and PSCs by disrupting the cancer-stromal interaction (P = 0.0002). Our findings indicate that calpeptin is a promising antitumor agent for pancreatic cancer, due not only to its suppressive effect on PCCs and PSCs but also its disruption of the cancer-stromal interaction.

Topics: Animals; Calpain; Cell Communication; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dipeptides; Disease Models, Animal; Gene Expression; Humans; Mice; Pancreatic Neoplasms; Pancreatic Stellate Cells; RNA, Messenger; Stromal Cells; Tumor Burden; Xenograft Model Antitumor Assays

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.

Topics: Animals; Calpain; Carbamates; Cell Survival; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Gene Knockdown Techniques; Humans; Inflammation; Microglia; Motor Neurons; Multiple Sclerosis; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Th1 Cells; Th17 Cells

The intracellular protein HMGB1 is released from cells and acts as a damage-associated molecular pattern molecule during many diseases, including inflammatory bowel disease (IBD); however, the intracellular function of HMGB1 during inflammation is poorly understood. Here, we demonstrated that cytosolic HMGB1 regulates apoptosis by protecting the autophagy proteins beclin 1 and ATG5 from calpain-mediated cleavage during inflammation. Colitis in mice with an intestinal epithelial cell-specific Hmgb1 deletion and patients with IBD were both characterized by increased calpain activation, beclin 1 and ATG5 cleavage, and intestinal epithelial cell (IEC) death compared with controls. In vitro cleavage assays and studies of enteroids verified that HMGB1 protects beclin 1 and ATG5 from calpain-mediated cleavage events that generate proapoptotic protein fragments. Together, our results indicate that HMGB1 is essential for mitigating the extent and severity of inflammation-associated cellular injury by controlling the switch between the proautophagic and proapoptotic functions of beclin 1 and ATG5 during inflammation. Moreover, these studies demonstrate that HMGB1 is pivotal for reducing tissue injury in IBD and other complex inflammatory disorders.

Topics: Adaptive Immunity; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Calpain; Cell Cycle Checkpoints; Cells, Cultured; Colitis; Cytosol; Dextran Sulfate; Dipeptides; Epithelial Cells; Female; HMGB1 Protein; Humans; Intestinal Mucosa; Male; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Proteolysis

Prion diseases are neurodegenerative disorders where infectious prion proteins (PrP) accumulate in brain leading to aggregation of amyloid fibrils and neuronal cell death. The amino acid sequence 106-126 from prion proteins, PrP(106-126), is highly amyloidogenic and implicated in prion-induced pathologies. As PrP is known to be expressed in blood following leakage from brain tissue, we sought to investigate its biological effects on human platelets, which have been widely employed as 'peripheral' model for neurons. Our findings suggested that, PrP(106-126) (20μM) induced dramatic 30-fold rise in intracellular calcium (from 105±30 to 3425±525nM) in platelets, which was attributable to influx from extracellular fluid with comparatively less contribution from intracellular stores. Calcium mobilization was associated with 8-10-fold stimulation in the activity of thiol protease calpain that led to partial cleavage of cytoskeleton-associated protein talin and extensive shedding of microparticles from platelets, thus transforming platelets to 'activated' phenotype. Both proteolysis of talin and microparticle release were precluded by calpeptin, a specific inhibitor of calpain. As microparticles are endowed with phosphatidylserine-enriched surface and hence are pro-coagulant in nature, exposure to prion favored a thrombogenic state in the organism.

Topics: Blood Coagulation; Blood Platelets; Calcium; Calcium Signaling; Calpain; Cell-Derived Microparticles; Cells, Cultured; Cytoskeleton; Dipeptides; Humans; Intracellular Space; Peptide Fragments; Platelet Activation; Prion Diseases; Prions; Proteolysis; Talin; Thrombosis

Aldosterone is a steroid hormone important in the regulation of blood pressure. Aberrant production of aldosterone results in the development and progression of diseases including hypertension and congestive heart failure; therefore, a complete understanding of aldosterone production is important for developing more effective treatments. Angiotensin II (AngII) regulates steroidogenesis, in part through its ability to increase intracellular calcium levels. Calcium can activate calpains, proteases classified as typical or atypical based on the presence or absence of penta-EF-hands, which are involved in various cellular responses. We hypothesized that calpain, in particular calpain-10, is activated by AngII in adrenal glomerulosa cells and underlies aldosterone production. Our studies showed that pan-calpain inhibitors reduced AngII-induced aldosterone production in 2 adrenal glomerulosa cell models, primary bovine zona glomerulosa and human adrenocortical carcinoma (HAC15) cells, as well as CYP11B2 expression in the HAC15 cells. Although AngII induced calpain activation in these cells, typical calpain inhibitors had no effect on AngII-elicited aldosterone production, suggesting a lack of involvement of classical calpains in this process. However, an inhibitor of the atypical calpain, calpain-10, decreased AngII-induced aldosterone production. Consistent with this result, small interfering RNA (siRNA)-mediated knockdown of calpain-10 inhibited aldosterone production and CYP11B2 expression, whereas adenovirus-mediated overexpression of calpain-10 resulted in increased AngII-induced aldosterone production. Our results indicate that AngII-induced activation of calpain-10 in glomerulosa cells underlies aldosterone production and identify calpain-10 or its downstream pathways as potential targets for the development of drug therapies for the treatment of hypertension.

Topics: Aldosterone; Angiotensin II; Animals; Calpain; Cattle; Cell Line, Tumor; Cells, Cultured; Dipeptides; Humans; Hydrocortisone; Zona Glomerulosa

Chronic alcohol consumption causes multifaceted damage to the central nervous system (CNS), underlying mechanisms of which are gradually being unraveled. In our previous studies, activation of calpain, a calcium-activated neutral protease has been found to cause detrimental alterations in spinal motor neurons following ethanol (EtOH) exposure in vitro. However, it is not known whether calpain plays a pivotal role in chronic EtOH exposure-induced structural damage to CNS in vivo. To test the possible involvement of calpain in EtOH-associated neurodegenerative mechanisms the present investigation was conducted in a well-established mouse model of alcohol dependence - chronic intermittent EtOH (CIE) exposure and withdrawal. Our studies indicated significant loss of axonal proteins (neurofilament light and heavy, 50-60%), myelin proteins (myelin basic protein, 20-40% proteolipid protein, 25%) and enzyme (2', 3'-cyclic-nucleotide 3'-phosphodiesterase, 21-55%) following CIE in multiple regions of brain including hippocampus, corpus callosum, cerebellum, and importantly in spinal cord. These CIE-induced deleterious effects escalated after withdrawal in each CNS region tested. Increased expression and activity of calpain along with enhanced ratio of active calpain to calpastatin (sole endogenous inhibitor) was observed after withdrawal compared to EtOH exposure. Pharmacological inhibition of calpain with calpeptin (25 μg/kg) prior to each EtOH vapor inhalation significantly attenuated damage to axons and myelin as demonstrated by immuno-profiles of axonal and myelin proteins, and Luxol Fast Blue staining. Calpain inhibition significantly protected the ultrastructural integrity of axons and myelin compared to control as confirmed by electron microscopy. Together, these findings confirm CIE exposure and withdrawal induced structural alterations in axons and myelin, predominantly after withdrawal and corroborate calpain inhibition as a potential protective strategy against EtOH associated CNS degeneration.

Topics: Administration, Inhalation; Alcoholism; Animals; Axons; Brain; Calpain; Central Nervous System Depressants; Dipeptides; Disease Models, Animal; Ethanol; Glycoproteins; Male; Mice, Inbred C57BL; Myelin Sheath; Nerve Degeneration; Neuroprotective Agents; Spinal Cord; Substance Withdrawal Syndrome

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, resulting in dopaminergic (DA) neuronal loss in the substantia nigra pars compacta (SNpc) and damage to the extranigral spinal cord neurons. Current therapies do not prevent the disease progression. Hence, developing efficacious therapeutic strategies for treatment of PD is of utmost importance. The goal of this study is to delineate the involvement of calpain-mediated inflammation and neurodegeneration in SN and spinal cord in MPTP-induced parkinsonian mice (C57BL/6 N), thereby elucidating potential therapeutic target(s). Increased calpain expression was found localized to tyrosine hydroxylase (TH(+)) neurons in SN with significantly increased TUNEL-positive neurons in SN and spinal cord neurons in MPTP mice. Inflammatory markers Cox-2, caspase-1, and NOS-2 were significantly upregulated in MPTP mouse spinal cord as compared to control. These parameters correlated with the activation of astrocytes, microglia, infiltration of CD4(+)/CD8(+) T cells, and macrophages. We found that subpopulations of CD4(+) cells (Th1 and Tregs) were differentially expanded in MPTP mice, which could be regulated by inhibition of calpain with the potent inhibitor calpeptin. Pretreatment with calpeptin (25 μg/kg, i.p.) attenuated glial activation, T cell infiltration, nigral dopaminergic degeneration in SN, and neuronal death in spinal cord. Importantly, calpeptin ameliorated MPTP-induced altered gait parameters (e.g., reduced stride length and increased stride frequency) as demonstrated by analyses of spatiotemporal gait indices using ventral plane videography. These findings suggest that calpain plays a pivotal role in MPTP-induced nigral and extranigral neurodegenerative processes and may be a valid therapeutic target in PD.

Topics: Animals; Astrocytes; Calpain; Dipeptides; Dopaminergic Neurons; Gait Disorders, Neurologic; Inflammation; Lymphocyte Subsets; Macrophages; Male; Mice; Mice, Inbred C57BL; Microglia; Nerve Degeneration; Nerve Tissue Proteins; Parkinsonian Disorders; Spinal Cord; Substantia Nigra

Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia-reperfusion injury. Isoforms of Ca(2+)-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia-reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia-reperfusion.. Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of >500μM of Ca(2+) to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca(2+)-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca(2+) to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30min of coronary occlusion followed by 30min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1.. We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart.

Topics: Animals; Blotting, Western; Calcium; Calpain; Dipeptides; Female; Humans; Male; Middle Aged; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Young Adult

Microvesicles shed from cells carry constituents of the cell cytoplasm, including, of importance in multidrug resistance to cancer chemotherapy, drugs that the tumor cell attempts to efflux. To see whether such drugs could be used at lower concentrations with the same efficacy, it was first shown that microvesiculation of prostate cancer (PCa) cells, PC3, could be inhibited pharmacologically with calpeptin (calpain inhibitor) and by siRNA (CAPNS1). In cells treated with docetaxel (DTX), this inhibition resulted in a third-fold increase in intracellular concentrations of DTX. As a result, 20-fold lower concentrations of DTX (5 nM) could be used, in the presence of calpeptin (20 μM) inducing the same degree of apoptosis after 48 h in PC3 cells, as 100 nM of DTX alone. Inhibition of microvesiculation similarly improved combination chemotherapy (DTX and methotrexate). In a mouse xenograft model of PCa, DTX (0.1 mg/kg) together with calpeptin (10 mg/kg), administered i.p., significantly reduced tumor volumes compared to DTX alone (0.1 mg/kg) and brought about the same reductions in tumor growth as 10 mg/kg of DTX alone. As well as further reducing vascularization, it also increased apoptosis and reduced proliferation of PC3 cells in tumor xenografts.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calpain; Cell Line, Tumor; Cell Proliferation; Cell-Derived Microparticles; Dipeptides; Docetaxel; Dose-Response Relationship, Drug; Drug Synergism; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Taxoids; Treatment Outcome

Acrylamide (ACR) is a vinyl monomer with established human neurotoxic effects, which is characterized by the accumulation of neurofilaments (NFs) in the distal swellings of large axons in peripheral and central nervous systems. However, the mechanisms of neurotoxicity remain unclear. The objective is to investigate the neuroprotective effect of calpeptin (CP) on ACR-induced neuropathy and its mechanism. Female adult Wistar rats were randomly divided into four groups (control, CP, ACR, and ACR + CP group). Control group received 0.9 % saline, ACR and ACR + CP groups received 30 mg/kg ACR by intraperitoneal injection. In addition, CP and ACR + CP groups also received 200 µg/kg CP. Gait analysis and hind limb splay were measured weekly to analyze neurobehavioral changes. The calpain activity and the changes of NFs protein levels in spinal cord are determined. Compared with control group, body weight of rats in ACR group decreased by 11.3 % (P < 0.01), while in ACR + CP group body weight increased significantly by 8.3 % (P < 0.01) compared with ACR group by the end of the 4th week; gait score of rats in both ACR and ACR + CP groups increased significantly by 167 % and 100 % (P < 0.01) compared with control group, while it decreased significantly by 25.1 % (P < 0.01) in ACR + CP group compared with ACR group; the distance of hind limb splay in both ACR and ACR + CP groups increased by 76.7 % and 49.5 % (P < 0.01) compared with control group, while it decreased by 15.4 % (P < 0.01) in ACR + CP group compared with ACR group; calpain activity of spinal cord at ACR and ACR + CP groups increased significantly by 14.9 % and 10.0 % (P < 0.01) compared with control group, while it decreased 4.2 % (P < 0.01) in ACR + CP group compared with ACR group; compared with control group, the levels of light NF (NF-L), medium NF (NF-M) and heavy NF (NF-H) subunits increased by 81.2 %, 263.6 % and 22.6 % (P < 0.01) in the supernatant of ACR group in spinal cord tissue and increased by 28.4 %, 96.6 % and 10.6 % (P < 0.01) in ACR + CP group, while the levels of NF-L, NF-M and NF-H subunits decreased by 29.1 %, 45.9 % and 9.8 % (P < 0.01) in ACR + CP group compared with ACR group. The present results suggested that CP can relieve ACR neuropathy by decrease calpain activity and NFs degradation. The changes of calpain activity and NFs may be one of the mechanisms of ACR-induced neuropathy.

Topics: Acrylamide; Animals; Behavior, Animal; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Female; Gait Disorders, Neurologic; Injections, Intraperitoneal; Neurofilament Proteins; Neuroprotective Agents; Neurotoxicity Syndromes; Rats; Rats, Wistar; Spinal Cord

Neoplastic cells accumulate magnesium, an event which provides selective advantages and is frequently associated with TRPM7 overexpression. Little is known about magnesium homeostasis in drug-resistant cancer cells. Therefore, we used the colon cancer LoVo cell model and compared doxorubicin-resistant to sensitive cells. In resistant cells the concentration of total magnesium is higher while its influx capacity is lower than in sensitive cells. Accordingly, resistant cells express lower amounts of the TRPM6 and 7, both involved in magnesium transport. While decreased TRPM6 levels are due to transcriptional regulation, post-transcriptional events are involved in reducing the amounts of TRPM7. Indeed, the calpain inhibitor calpeptin markedly increases the levels of TRPM7 in resistant cells. In doxorubicin-sensitive cells, silencing TRPM7 shifts the phenotype to one more similar to resistant cells, since in these cells silencing TRPM7 significantly decreases the influx of magnesium, increases its intracellular concentration and increases resistance to doxorubicin. On the other hand, calpain inhibition upregulates TRPM7, decreases intracellular magnesium and enhances the sensitivity to doxorubicin of resistant LoVo cells. We conclude that in LoVo cells drug resistance is associated with alteration of magnesium homeostasis through modulation of TRPM7. Our data suggest that TRPM7 expression may be an additional undisclosed player in chemoresistance.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Calpain; Cell Line, Tumor; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dipeptides; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Ion Transport; Magnesium; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; TRPM Cation Channels

CTEN/TNS4 is a member of the Tensin gene family. It localizes to focal adhesions and induces cell motility. The mechanisms regulating Cten expression are unclear although we have shown regulation by Kras in the colon and pancreas. In normal mammary cell lines, it is reportedly upregulated by epidermal growth factor receptor (EGFR) and STAT3 signalling and upregulation is accompanied by downregulation of Tensin 3 (Tensin switch). In this study, we investigated the roles of EGFR and STAT3 signalling in the regulation of Cten in colorectal cancer (CRC). In addition, we investigated calpain--a regulator of focal adhesion-associated proteins whose relevance to Cten has not been investigated. CRC cell lines were stimulated with epidermal growth factor (EGF). This resulted in an increase in Cten and Tensin 3 protein. Kras was knocked down and this resulted in downregulation of Cten and Tensin 3. We next investigated the role of STAT3 signalling. Activation and knockdown of STAT3 resulted in downregulation and upregulation, respectively, of Cten. Inhibition of calpain resulted in upregulation of both Cten and Tensin 3. As the regulators of Cten also seemed to regulate Tensin 3, we tested the interaction between Cten and Tensin 3. Cten was forcibly expressed or knocked down resulting, respectively, in upregulation and downregulation of Tensin 3. We conclude that in CRC, Cten is upregulated by EGFR and Kras but downregulated by STAT3. We show that calpain may be a negative regulator of Cten and that a Tensin switch does not occur and, if anything, Cten stabilizes Tensin 3.

Topics: Calpain; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HCT116 Cells; Humans; Microfilament Proteins; Protein Stability; Proto-Oncogene Proteins p21(ras); Signal Transduction; STAT3 Transcription Factor; Tensins; Transfection

Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca(2+) and leads to activation of calpain, a Ca(2+)-dependent cysteine protease. We have shown previously that S-allyl-L-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, L-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of μ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5 pM), which blocks the active site of calpain, and calpastatin (100 pM), an endogenous calpain-inhibitor protein, additively inhibited μ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25 μM), which prevents interaction of Ca(2+) with the Ca(2+)-binding site of calpain, was unaffected by SAC. In contrast, SAC (1 mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100 μM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of μ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca(2+)-binding site.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell Survival; Cells, Cultured; Cysteine; Dipeptides; Endoplasmic Reticulum Stress; Hippocampus; Leupeptins; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Wistar; Spectrin

Understanding the molecular mechanism of prostate cancer progression from androgen dependence to independence may lead to developing more effective treatments against prostate cancer. Herein, our previous in vitro model was employed to assess the effects of continuous androgen-deprivation on developing the metastatic phenotype from androgen-dependent prostate cancer cells (LNCaP). The results indicated that long-term androgen deprivation resulted in overexpression of calpain 2 and increased expression of filamin A (FlnA), but not for calpain 1. The enhanced activity of calpain 2 was confirmed by the accumulation of cleaved FlnA fragments, which could be effectively blocked by calpeptin (an inhibitor of calpain 2). Therefore, the combination of calpain 2 inhibitor and androgen deprivation may provide new therapeutic strategy for patients to prevent or postpone prostate cancer progression.

Topics: Androgens; Blotting, Western; Calpain; Dipeptides; Filamins; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Male; Microscopy, Confocal; Prostatic Neoplasms; Receptors, Androgen; Tumor Cells, Cultured

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.

Topics: Blood Platelets; Blotting, Western; Calpain; Cells, Cultured; Dipeptides; Estrenes; Flow Cytometry; Gallic Acid; Humans; Immunoprecipitation; Male; Myosins; Protein Transport; Pyrrolidinones; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Signal Transduction; Thrombin; Toll-Like Receptor 4

Deficiency in energy metabolisms is perhaps the earliest modifiable defect in brain aging and sporadic Alzheimer's disease (sAD). Several high-energy compounds (HECs) such as ATP, phosphoenolpyruvate, phosphocreatine and acetyl coenzyme A have been shown to exhibit neuroprotective effects. To understand their mechanism of actions, we tested the effects of these HECs on intracellular Ca(2+), a central regulator in brain function. Our data showed that the HECs robustly and dose-dependently mobilized intracellular Ca(2+) in cultured SH-SY5Y cells, and the actions were sensitive to intracellular Ca(2+) chelator BAPTA-AM or energy metabolism blocker rotenone. The Ca(2+) influx triggered by the HECs was from both extracellular medium and intracellular stores and the HECs also induced repetitive Ca(2+) oscillations. As these actions were similar to those of classical Ca(2+) agonists, the HECs may be viewed as a new group of physiological Ca(2+) agonists. We also found that the HECs promoted the intracellular activity of calpain, a Ca(2+)-dependent protease, and the enzyme activity fluctuated in concert with cellular energy levels, suggesting that calpain activity may also be energy-driven or energy-dependent. These findings may add to current knowledge for the regulatory mechanisms of Ca(2+) and calpain. Since Ca(2+) and calpain undergo critical dysfunction in brain aging but the underlying mechanisms remain elusive, our work may provide a new perspective for clarifying some controversies. More importantly, the HECs, as key intermediates in glucose catabolism, the primary source of energy supply in the brain, may be used as potential drugs for rational prevention of sAD.

Topics: Acetyl Coenzyme A; Adenosine Triphosphate; Alzheimer Disease; Calcium; Calpain; Cell Line, Tumor; Chelating Agents; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Extracellular Space; Glutamic Acid; Humans; Intracellular Space; Nicotine; Nicotinic Agonists; Phosphocreatine; Phosphoenolpyruvate; Rotenone; Uncoupling Agents

Interleukin (IL)-17A plays an important role in the cerebral ischemia/reperfusion (I/R) injury. However, the mechanisms are still largely unknown. Calpain-transient receptor potential canonical (subtype) 6 (TRPC6) signaling pathway has been recently found to be implicated in brain I/R injury. However, their relationships with IL-17A remain unknown. This study aims to test whether this important signaling has correlation with IL-17A and how they led to the neuronal damage in I/R injury. In the present study, mice were subjected to middle cerebral artery occlusion (60 min) followed by reperfusion for different times. Infarct volumes and neurological deficits were examined. Real-time PCR (RT-PCR) and Western blotting were conducted to detect IL-17A expression in the penumbral brain tissue. Activation of calpain and expression of TRPC6 were also studied. We found that cerebral I/R significantly increased the levels of IL-17A at 1, 3 and 6 days after reperfusion in the penumbral area. IL-17A knockout or anti-IL-17A monoclonal antibody (mAb) significantly reduced whereas recombinant mouse-IL-17A (rIL-17A) increased the activation of calpain at 3 days after reperfusion. The calpain specific inhibitor calpeptin significantly increased TRPC6 expression. Brain injury and neurological deficits were largely abrogated by IL-17A knockout, anti-IL-17A mAb or calpeptin. Recombinant IL-17A treatment markedly increased I/R injury. In conclusion, IL-17A may promote brain I/R injury through the increase of calpain-mediated TRPC6 proteolysis. These results further outline a novel neuroprotective strategy with increased effectiveness for the inhibition of excess brain IL-17A in cerebral I/R injury.

Topics: Animals; Brain; Brain Ischemia; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Infarction, Middle Cerebral Artery; Interleukin-17; Male; Mice, Inbred C57BL; Mice, Knockout; Proteolysis; Recombinant Proteins; Reperfusion Injury; Time Factors; TRPC Cation Channels; TRPC6 Cation Channel

To investigate the effect of calpeptin on diaphragmatic injury and atrophy under controlled mechanical ventilation in rats.. A total of 24 SPF Sprague-Dawley (SD) rats were randomly divided into anesthetized control group (CON group), 24-hour controlled mechanical ventilation group (CMV group), and 24-hour CMV + treatment with calpeptin group (CMVC group), with 8 rats in each group. Animals in the CON group received an intraperitoneal injection of pentobarbital sodium without CMV and continuous infusion of pentobarbital sodium. A small-animal ventilator was used for 24 hours in rats of CMV group. Rats of CMVC were treated with a specific calpain inhibitor calpeptin (4 mg/kg). The drug was injected subcutaneously 2 hours before and 8, 15 and 23 hours after mechanical ventilation. Changes in diaphragm ultrastructure, light microscopic picture, and myosin heavy chain (MHC) expression were observed.. (1) Alignment of myofilaments and normal Z-band, and the shape of mitochondria were maintained in CON group as revealed by electron microscope. The signs of misalignment of myofibrils, disruption of Z-band and vacuolar mitochondria were found in CMV group, and they were obviously improved in CMVC group. The density of muscle injury (× 10⁻²/μm²) in CMV group was significantly higher than that in control group (36.8 ± 13.7 vs. 6.4 ± 6.3, t=6.373, P=0.001), and that in CMVC group was significantly lowered (17.6 ± 9.1 vs. 36.8 ± 13.7, t=3.694, P=0.002).(2) In CON group, the diaphragm fibers appeared regular in cross section without pathologic change under light microscopy. Fuzzy muscle striations, irregular muscle fibers, centralized nuclei and swelling of capillary endothelial cells were observed in CMV group, while pathological changes in the CMVC group were milder significantly. (3) In CMV group, the density of MHCslow and MHCfast was lower compared with that of CON group, and the gray value was lowered by 61.1% (t=8.138, P=0.001) and 77.1% (t=8.844, P=0.001), respectively, especially in MHCfast. However, the gray values of MHCslow and MHCfast were increased by 1.51 folds (t=4.601, P=0.010), and 1.33 folds (t=2.859, P=0.011), respectively, after treatment with calpeptin, and the elevation was more significantly in MHCslow.. Diaphragmatic injury and atrophy were found after CMV for 24 hours. Calpeptin could reverse the detrimental effects of CMV, and it suggested that calpain plays an important role in modulating the ventilator-induced dysfunction of the diaphragm.

Topics: Animals; Atrophy; Calpain; Diaphragm; Dipeptides; Disease Models, Animal; Male; Myosin Heavy Chains; Rats; Rats, Sprague-Dawley; Respiration, Artificial

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation.. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling.. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Topics: Animals; Anoctamins; Blood Platelets; Calcium Signaling; Calpain; CD36 Antigens; Cell Membrane; Crotalid Venoms; Dipeptides; Humans; Lectins, C-Type; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Phosphatidylserines; Phospholipid Transfer Proteins; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Structure, Quaternary; Proteolysis; src-Family Kinases; Talin; Thrombin

Wiskott-Aldrich syndrome protein (WASP) links T-cell receptor (TCR) signaling to the actin cytoskeleton. WASP is normally protected from degradation by the Ca(++)-dependent protease calpain and by the proteasome because of its interaction with the WASP-interacting protein.. We investigated whether WASP is degraded after TCR ligation and whether its degradation downregulates F-actin assembly caused by TCR ligation.. Primary T cells, Jurkat T cells, and transfected 293T cells were used in immunoprecipitation experiments. Intracellular F-actin content was measured in splenic T cells from wild-type, WASP-deficient, and c-Casitas B-lineage lymphoma (Cbl)-b-deficient mice by using flow cytometry. Calpeptin and MG-132 were used to inhibit calpain and the proteasome, respectively.. A fraction of WASP in T cells was degraded by calpain and by the ubiquitin-proteasome pathway after TCR ligation. The Cbl-b and c-Cbl E3 ubiquitin ligases associated with WASP after TCR signaling and caused its ubiquitination. Inhibition of calpain and lack of Cbl-b resulted in a significantly more sustained increase in F-actin content after TCR ligation in wild-type T cells but not in WASP-deficient T cells.. TCR ligation causes WASP to be degraded by calpain and to be ubiquitinated by Cbl family E3 ligases, which targets it for destruction by the proteasome. WASP degradation might provide a mechanism for regulating WASP-dependent TCR-driven assembly of F-actin.

Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Calpain; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Down-Regulation; Leupeptins; Mice; Mice, Knockout; Proto-Oncogene Proteins c-cbl; Receptors, Antigen, T-Cell; T-Lymphocytes; Ubiquitination; Wiskott-Aldrich Syndrome Protein

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Topics: Acrylates; Animals; Bacterial Toxins; Blotting, Western; Calcium; Calpain; Cell Membrane; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Endothelial Cells; Flow Cytometry; HMGB1 Protein; Imidazoles; Indoles; Intracellular Space; Ion Transport; L-Lactate Dehydrogenase; Microscopy, Electron, Transmission; Necrosis; Potassium; Swine

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Recently, the neurodegenerative component of multiple sclerosis has come under focus particularly because permanent disability in patients correlates well with neurodegeneration; and observations in both humans and multiple sclerosis animal models highlight neurodegeneration of retinal ganglion cells as an early event. After myelin oligodendrocyte glycoprotein immunization of Brown Norway rats, significant retinal ganglion cell loss precedes the onset of pathologically defined autoimmune optic neuritis. To study the role calcium and calpain activation may play in mediating early degeneration, manganese-enhanced magnetic resonance imaging was used to monitor preclinical calcium elevations in the retina and optic nerve of myelin oligodendrocyte glycoprotein-immunized Brown Norway rats. Calcium elevation correlated with an increase in calpain activation during the induction phase of optic neuritis, as revealed by increased calpain-specific cleavage of spectrin. The relevance of early calpain activation to neurodegeneration during disease induction was addressed by performing treatment studies with the calpain inhibitor calpeptin. Treatment not only reduced calpain activity but also protected retinal ganglion cells from preclinical degeneration. These data indicate that elevation of retinal calcium levels and calpain activation are early events in autoimmune optic neuritis, providing a potential therapeutic target for neuroprotection.

Topics: Amyloid beta-Protein Precursor; Animals; Calcium; Calpain; Chlorides; Dipeptides; Disease Models, Animal; Ectodysplasins; Encephalomyelitis, Autoimmune, Experimental; Female; Magnetic Resonance Imaging; Manganese Compounds; Myelin-Oligodendrocyte Glycoprotein; Nerve Degeneration; Neuroprostanes; Optic Nerve; Optic Neuritis; Rats; Retina; Retinal Ganglion Cells; Time Factors

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Arsenite Transporting ATPases; Calpain; Cell Death; Cell Line; Dipeptides; Dopaminergic Neurons; Electrophoresis, Gel, Two-Dimensional; Glycine; Infarction, Middle Cerebral Artery; Ionomycin; Peripherins; Proteome; Proteomics; Rats; Rats, Sprague-Dawley

Optic neuritis (ON), which is an acute inflammatory autoimmune demyelinating disease of the central nervous system (CNS), often occurs in multiple sclerosis (MS). ON is an early diagnostic sign in most MS patients caused by damage to the optic nerve leading to visual dysfunction. Various features of both MS and ON can be studied following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in Lewis rats. Inflammation and cell death in the optic nerve, with subsequent damage to the retinal ganglion cells in the retina, are thought to correlate with visual dysfunction. Thus, characterizing the pathophysiological changes that lead to visual dysfunction in EAE animals may help develop novel targets for therapeutic intervention. We treated EAE animals with and without the calpain inhibitor calpeptin (CP). Our studies demonstrated that the Ca(2+)-activated neutral protease calpain was upregulated in the optic nerve following induction of EAE at the onset of clinical signs (OCS) of the disease, and these changes were attenuated following treatment with CP. These reductions correlated with decreases in inflammation (cytokines, iNOS, COX-2, and NF-κB), and microgliosis (i.e. activated microglia). We observed that calpain inhibition reduced astrogliosis (reactive astroglia) and expression of aquaporin 4 (AQP4). The balance of Th1/Th2 cytokine production and also expression of the Th1-related CCR5 and CXCR3 chemokine receptors influence many pathological processes and play both causative and protective roles in neuron damage. Our data indicated that CP suppressed cytokine imbalances. Also, Bax:Bcl-2 ratio, production of tBid, PARP-1, expression and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated after treatment with CP. Our results demonstrated that CP decreased demyelination [loss of myelin basic protein (MBP)] and axonal damage [increase in dephosphorylated neurofilament protein (de-NFP)], and also promoted intracellular neuroprotective pathways in optic nerve in EAE rats. Thus, these data suggest that calpain is involved in inflammatory as well as in neurodegenerative aspects of the disease and may be a promising target for treating ON in EAE and MS.

Topics: Animals; Apoptosis; Aquaporin 4; Calcium; Calpain; Cyclooxygenase 2; Cytokines; Dipeptides; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gliosis; Glycoproteins; Male; Molecular Weight; Myelin Basic Protein; NF-kappa B; Nitric Oxide Synthase Type II; Optic Nerve; Optic Neuritis; Rats; Rats, Inbred Lew; Signal Transduction

Studies on skeletal muscle cell specification and development have demonstrated in the past that calpains interact with various transcriptional factors in regulating the cellular function. It has therefore, been assumed that transcriptional factors like myogenin, MyoD, Myf5, and MRF4 that are active during the myogenic differentiation might be affected and degraded by calpains. Therefore, to examine the biochemical adaptations of myoblasts during myocyte formation and muscle development comprehensively, the current study was designed to identify the effect of calpeptin (calpain inhibitors) on protein expression during differentiation of C2C12 mouse myoblast. Cells were proliferated to near 80% confluence under Dulbecco's modified eagle medium and differentiated further in 2% HS with 50 μM calpeptin. Incubated cells were collected at 0, 12, and 72 h and later the cell proteins were focused onto pH 4-7 IEF strip, followed by 12.5% SDS-PAGE. Obtained spots on the gels were compared and matched using commercial 2-DE analysis software and matched spots were identified by MALDI-ToF and/or Q-Tof systems. Conclusively, cell differentiation was observed to be active from 12 to 72 h however, calpeptin affected the differentiation process and cut down the rate of fusion by approximately 50%. Out of 41 proteins identified, 12 proteins were found to be upregulated where as 29 proteins were downregulated.

Topics: Animals; Calpain; Cell Differentiation; Dipeptides; Gene Expression Regulation, Developmental; Mice; Muscle Development; Myoblasts; Protein Interaction Maps; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Cell Line, Tumor; Cells, Cultured; Dipeptides; Excitatory Amino Acid Agonists; Female; Hippocampus; Humans; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; N-Methylaspartate; Organ Culture Techniques; Pregnancy

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

Topics: Animals; Astrocytes; Biomarkers; Calcium; Calcium-Binding Proteins; Calpain; Cell Count; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Proliferation; Dipeptides; Gene Silencing; Intermediate Filament Proteins; Mice; Nerve Tissue Proteins; Nestin; Neural Stem Cells; Neurogenesis; Neurons; Phenotype; Tubulin

The USA300 strains of Staphylococcus aureus are the major cause of skin and soft tissue infection in the United States. Invasive USA300 infection has been attributed to several virulence factors, including protein A and the α-hemolysin (Hla), which cause pathology by activating host signaling cascades. Here we show that S. aureus exploits the proinflammatory bias of human keratinocytes to activate pyroptosis, a caspase 1-dependent form of inflammatory cell death, which was required for staphylococci to penetrate across a keratinocyte barrier. Keratinocyte necrosis was mediated by calpains, Ca(2+)-dependent intracellular proteases whose endogenous inhibitor, calpastatin, is targeted by Hla-induced caspase 1. Neither Panton-Valentine leukocidin nor protein A expression was essential, but inhibition of either calpain or caspase 1 activity was sufficient to prevent staphylococcal invasion across the keratinocytes. These studies suggest that pharmacological interruption of specific keratinocyte signaling cascades as well as targeting the Hla might prevent invasive skin infection by staphylococci.

Topics: Apoptosis; Calcium-Binding Proteins; Calpain; Caspase 1; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Humans; Keratinocytes; Methicillin-Resistant Staphylococcus aureus; Mutation; Signal Transduction; Soft Tissue Infections; Staphylococcal Infections; Staphylococcal Skin Infections; Virulence Factors

HD-PTP (PTPN23) is a non-transmembrane protein tyrosine phosphatase which contributes to the signal transduction pathways involved in the regulation of cell migration and invasion. We here demonstrate in T24 bladder carcinoma cells that HD-PTP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. In addition, treatment of the cells with the calpain inhibitor calpeptin results in the redistribution of endogenous HD-PTP to the periphery of the cells. Since (i) calpains are overexpressed in some tumors and (ii) the downregulation of HD-PTP enhances cell migration and invasion, we propose that HD-PTP degradation by calpains might result in the acquisition of a more aggressive phenotype in neoplastic cells.

Topics: Calcium; Calpain; Cell Line, Tumor; Cell Movement; Cysteine Proteinase Inhibitors; Dipeptides; Humans; Neoplasm Invasiveness; Protein Tyrosine Phosphatases, Non-Receptor; Proteolysis

Berbamine, a natural compound from Barberry, was reported to protect myocardium from ischemia/reperfusion (I/R) injury, but the underlying mechanisms are largely unknown.. Berbamine pretreatment from 10 to 100nmol/L concentration-dependently improved post-ischemic myocardial function. Similar protection was confirmed in isolated cardiomyocytes characterized by the attenuation of I/R-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) overloading and the depression of cell shortening and Ca(2+) transients, which were partially mimicked but not augmented by calpain inhibitor calpeptin and abolished by mitochondrial ATP-sensitive potassium (mitoK(ATP) channel inhibitor 5-hydroxydecanoate (5-HD) and phosphoinositide 3-kinase (PI3K) inhibitor wortmannin. Consistently, I/R-induced increase of calpain activity and decrease of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) activity; and protein expression of SERCA2a, desmin, calpastatin and Akt was significantly attenuated by berbamine. In addition, I/R-decreased Akt protein was reversed by calpeptin. Moreover, berbamine further increased I/R-enhanced phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β). These protections were abolished by wortmannin. Furthermore, berbamine significantly attenuated I/R-induced lactate dehydrogenase release, infarct size and contractile dysfunction, and such cardioprotective actions were abolished by wortmannin and 5-HD or mimicked by glycogen synthase kinase-3β (GSK3β) inhibitor SB216763 but without additive effect.. These findings suggest that berbamine confers cardioprotection against I/R injury by attenuating [Ca(2+)inf(i) overloading and preventing calpain activation through the activation of the PI3K-Akt-GSK3β pathway and, subsequently, opening of the mitoK(ATP) channel.

Topics: Androstadienes; Animals; Anti-Arrhythmia Agents; Benzylisoquinolines; Calcium; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Male; Muscle Proteins; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Wortmannin

Muscle weakness and atrophy are important manifestations of multiple sclerosis (MS). To investigate the pathophysiological mechanisms of skeletal muscle change in MS, we induced experimental autoimmune encephalomyelitis (EAE) in Lewis male rats and examined morphological and molecular changes in skeletal muscle. We also treated EAE rats with calpepetin, a calpain inhibitor, to examine its beneficial effects on skeletal muscle damage. Morphological changes in muscle tissue of EAE rats included smaller and irregularly shaped muscle fibers and fibrosis. Western blot analysis demonstrated increased calpain:calpastatin ratio, inflammation-related transcription factors (nuclear factor-κB:inhibitor of κB α ratio), and proinflammatory enzymes (cyclooxygenase-2). TUNEL-positive myonuclei in skeletal muscle cells of EAE rats indicated cell death. In addition, markers of apoptotic cell death (Bax:Bcl-2 ratio and caspase-12 protein levels) were elevated. Expression of muscle-specific ubiquitin ligases (muscle atrophy F-box and muscle ring finger protein 1), was upregulated in muscle tissue of EAE-vehicle animals. Both prophylactic and therapeutic treatment with calpeptin partially attenuated muscle changes noted in EAE animals. These results indicate that morphological and molecular changes including apoptotic cell death and protein breakdown develop in skeletal muscle of EAE animals and that these changes can be reversed by calpain inhibition.

Topics: Animals; Apoptosis; Blotting, Western; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Encephalomyelitis, Autoimmune, Experimental; In Situ Nick-End Labeling; Male; Muscle, Skeletal; Rats; Rats, Inbred Lew

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 previously shown that the cultured L6 myoblasts are susceptible to menadione-induced oxidative stress. Damaged cells were detached from the culture dishes. In the present study, we focused on focal adhesion kinase (FAK), which plays pivotal roles in maintaining focal adhesion function and cell survival. FAK, normally localized at the focal adhesion regions of the myoblasts, was not observed at the regions under oxidative stress induced by menadione and H(2) O(2) . Two cleavage products, 80-kDa N-terminal FAK and 35-kDa C-terminal FAK fragments, as well as full-length FAK (125 kDa) were detected in myoblasts cultured under normal conditions by western blotting with anti-N-terminal FAK or anti-C-terminal FAK sera. Of interest was the finding that the cleavage products of FAK (but not full-length FAK) disappeared under oxidative stress. The cleavage of full-length FAK to N-terminal FAK and C-terminal FAK was inhibited by calpeptin, a specific calpain inhibitor. In addition, pre-incubation of cells with calpeptin resulted in a sharp decrease in survival signals, such as Akt phosphorylation and the ratio of Bcl-2/Bax, under stress conditions. By contrast, not only relative viability, but also Akt phosphorylation and the ratio of Bcl-2/Bax was significantly improved when cells were transfected with a DNA construct of N-terminal FAK-Myc. These results suggest that the N-terminal FAK positively regulates survival signalling in early phases of oxidative stress in the cultured myoblasts.

Topics: Animals; Apoptosis; Blotting, Western; Calpain; Cell Adhesion; Cell Survival; Cells, Cultured; Dipeptides; Focal Adhesion Protein-Tyrosine Kinases; Mice; Myoblasts; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

In idiopathic inflammatory myopathies (IIMs), extracellular inflammatory stimulation is considered to induce secondary intracellular inflammatory changes including expression of major histocompatibility complex class-I (MHC-I) and to produce a self-sustaining loop of inflammation. We hypothesize that activation of calpain, a Ca(2+) -sensitive protease, bridges between these extracellular inflammatory stress and intracellular secondary inflammatory changes in muscle cells. In this study, we demonstrated that treatment of rat L6 myoblast cells with interferon-γ (IFN-γ) caused expression of MHC-I and inflammation-related transcription factors (phosphorylated-extracellular signal-regulated kinase 1/2 and nuclear factor-κB). We also demonstrated that treatment with tumor necrosis factor-α (TNF-α) induced apoptotic changes and activation of calpain and cyclooxygenase-2. Furthermore, we found that posttreatment with calpeptin attenuated the intracellular changes induced by IFN-γ or TNF-α. Our results indicate that calpain inhibition attenuates apoptosis and secondary inflammatory changes induced by extracellular inflammatory stimulation in the muscle cells. These results suggest calpain as a potential therapeutic target for treatment of IIMs.

Topics: Animals; Apoptosis; Blotting, Western; Calpain; Cell Line; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Dipeptides; Histocompatibility Antigens Class I; Inflammation; Interferon-gamma; Myoblasts; Myositis; NF-kappa B; Rats; Signal Transduction; Transcription, Genetic; Tumor Necrosis Factor-alpha

The ATP-binding cassette transporter ABCG1 mediates cholesterol efflux from macrophages, and prevents the progression of macrophage foam-cell formation. Much less is known about the regulatory mechanism of ABCG1, although its physiological importance is becoming clearer. Here, we show the role of calpain in ABCG1 degradation.. Purified μ-calpain cleaved ABCG1 in crude membrane fractions prepared from human ABCG1-expressing HEK293 (ABCG1-HEK) cells. In ABCG1-HEK cells, calpeptin treatment, a calpain inhibitor, inhibited ABCG1 degradation, and thereby increased the expression and cholesterol efflux function of ABCG1. Biotinylation study demonstrated greater ABCG1 induction with calpeptin treatment in cell surface than that in whole cell lysates. Together with the result that increased ABCG1 expression with calpeptin treatment was observed under clathrin heavy-chain (CHC) knockdown conditions, where ABCG1 internalization was prevented, calpain is considered to catalyze ABCG1 cleavage on the plasma membrane. In mouse peritoneal macrophages as well as in ABCG1-HEK cells, calpeptin treatment inhibited ABCG1 degradation and enhanced ABCG1 expression, even under CHC-depleted conditions.. These observations indicate that calpain promotes ABCG1 degradation by cleaving cell surface-resident ABCG1, and consequently reduces the expression and cholesterol efflux function of ABCG1. Inhibition of ABCG1 cleavage by calpain could be a novel approach to suppress the progression of atherosclerosis.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; Calpain; Dipeptides; HEK293 Cells; Humans; Lipoproteins; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 μM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 μM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Calpain; Camptothecin; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Dipeptides; DNA Damage; DNA-Binding Proteins; Humans; Morpholines; Neuroblastoma; Protein Serine-Threonine Kinases; Purines; Pyrones; Roscovitine; Tumor Suppressor Proteins

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca(2+)](i)) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca(2+)-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca(2+)](i) increase in OV2008 cells was not effected by the elimination of extracellular Ca(2+), but this was attenuated by the depletion of internal Ca(2+) store, indicating that mobilization of intracellular Ca(2+]) stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca(2+)-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca(2+)/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Calcium; Calpain; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Dipeptides; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Humans; Immunoprecipitation; Intracellular Space; Nuclear Proteins; Ovarian Neoplasms; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Tumor Protein p73; Tumor Suppressor Proteins

Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with the pathogenesis of multiple sclerosis (MS) and is initiated by the attack of autoreactive T cells against self-myelin antigens, resulting in demyelination, degeneration of retinal ganglion cells (RGCs), and cumulative visual impairment.. Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats on day 0, and animals received daily intraperitoneal injections of calpain inhibitor (calpeptin) or vehicle from day 1 until killed. Retinal cell death was analyzed by DNA fragmentation, and surviving ganglion cells were quantified after double labeling of retinal tissue with TUNEL and Brn3a. The expression of apoptotic and inflammatory proteins was determined by Western blotting.. It was demonstrated that calpain inhibition downregulates expression of proapoptotic proteins and the proinflammatory molecule nuclear factor-kappa B (NF-κB) in the retina of Lewis rats with acute EAE. Immunofluorescent labeling revealed that apoptotic cells in the RGC layer of vehicle-treated EAE animals were Brn3a positive, and a moderate dose of calpeptin dramatically reduced the frequency of apoptotic RGCs.. These results suggest that calpain inhibition might be a useful supplement to immunomodulatory therapies such as corticosteroids in ON, due to its neuroprotective effect on RGCs.

Topics: Acute Disease; Animals; Apoptosis; Blotting, Western; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Gene Expression Regulation; In Situ Nick-End Labeling; Injections, Intraperitoneal; Male; Optic Neuritis; Rats; Rats, Inbred Lew; Retinal Ganglion Cells; RNA; Treatment Outcome

The dysfunction of the proteasome system is implicated in the pathomechanism of several chronic neurodegenerative diseases. Lactacystin (LC), an irreversible proteasome inhibitor, induces cell death in primary cortical neurons, however, the molecular mechanisms of its neurotoxic action has been only partially unraveled. In this study we aimed to elucidate an involvement of the key enzymatic pathways responsible for LC-induced neuronal cell death. Incubation of primary cortical neurons with LC (0.25-50 μg/ml) evoked neuronal cell death in concentration- and time-dependent manner. Lactacystin (2.5 μg/ml; 6.6μM) enhanced caspase-3 activity, but caspase-3 inhibitor, Ac-DEVD-CHO did not attenuate the LC-evoked cell damage. Western blot analysis showed a time-dependent, prolonged activation of MAPK/ERK1/2 pathway after LC exposure. Moreover, inhibitors of MAPK/ERK1/2 signaling, U0126 and PD98052 attenuated the LC-evoked cell death. We also found that LC-treatment resulted in the induction of calpains and calpain inhibitors (MDL28170 and calpeptin) protected neurons against the LC-induced cell damage. Neuroprotective action of MAPK/ERK1/2 and calpain inhibitors were connected with attenuation of LC-induced DNA fragmentation measured by Hoechst 33342 staining and TUNEL assay. However, only MAPK/ERK1/2 but not calpain inhibitors, attenuated the LC-induced AIF (apoptosis inducing factor) release. Further studies showed no synergy between neuroprotective effects of MAPK/ERK1/2 and calpain inhibitors given in combination when compared to their effects alone. The obtained data provided evidence for neuroprotective potency of MAPK/ERK1/2 and calpain, but not caspase-3 inhibition against the neurotoxic effects of LC in primary cortical neurons and give rationale for using these inhibitors in the treatment of neurodegenerative diseases connected with proteasome dysfunction.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Butadienes; Calpain; Caspase 3; Caspase Inhibitors; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytoprotection; Dipeptides; Dose-Response Relationship, Drug; Enzyme Activation; Gestational Age; In Situ Nick-End Labeling; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Neuroprotective Agents; Nitriles; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Signal Transduction; Spectrin; Time Factors

Parkinson's disease (PD), characterized by selective midbrain nigrostriatal dopaminergic degeneration, is consistently associated with moderate systemic mitochondrial dysfunction. Downstream degeneration of spinal cord has also been suggested in PD, although the mechanisms have not been much investigated. In the present study, two mitochondrial toxicants, 1-methyl-4-phenylpyridinium ion (MPP(+)) and rotenone were tested in ventral spinal cord (VSC 4.1) motoneuronal cells. Cell death was assessed by morphological and biochemical means to discern a lower apoptosis-inducing concentration and lethal concentration of 50% cell death (LC(50)), which were subsequently compared in further cytoprotection experiments. Mitochondrial toxicants dose-dependently induced increase in intracellular free Ca(2+) level, which was conducive for increased expression and activities of Ca(2+)-activated neutral protease calpain and downstream caspase-3. Thus, mitochondrial damage triggered apoptotic mechanisms in spinal cord motoneurons. Inhibition of calpain by calpeptin significantly attenuated damaging effects of MPP(+) and rotenone on motoneurons, especially at low apoptosis-inducing concentrations of toxicants and partly at their LC(50), as demonstrated by absence of DNA ladder formation and decrease in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Cytoprotection by calpeptin was observed with marked decreases in Bax: Bcl-2 ratio and activities of calpain and caspase-3, which affirmed the role of mitochondrial dysfunction and involvement of intrinsic pathway in mediation of apoptosis. These findings strongly suggested that parkinsonian toxicants MPP(+) and rotenone at low doses induced cascade of cell-damaging effects in spinal cord motoneurons, thus, highlighting the possibility of induction of apoptotic mechanisms in these cells, when subjected to mitochondrial stress. Cytoprotection rendered by calpeptin further validated the involvement of calpain in apoptosis and suggested calpain inhibition as a potential neuroprotective strategy.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Blotting, Western; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Hybrid Cells; In Situ Nick-End Labeling; Mice; Motor Neurons; Rotenone; Spinal Cord; Uncoupling Agents

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and µ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and µ-calpain may be involved in colon epithelial cell death induced by E. histolytica.

Topics: Calpain; Cell Death; Cell Line; Cell Survival; Dipeptides; Entamoeba histolytica; Epithelial Cells; Gene Knockdown Techniques; Humans

The reduced regenerative potential of muscle fibres, most likely due to a decreased number and/or function of satellite cells, could play a significant role in the progression of muscle ageing. Accumulation of reactive oxygen species has been clearly correlated to sarcopenia and could contribute to the impairment of satellite cell function. In this work we have investigated the effect of oxidative stress generated by hydrogen peroxide in cultured human skeletal muscle satellite cells. We specifically focused on the activity and regulation of calpains. These calcium-dependent proteases are known to regulate many transduction pathways including apoptosis and play a critical role in satellite cell function. In our experimental conditions, which induce an increase in calcium concentration, protein oxidation and apoptotic cell death, a significant up-regulation of calpain expression and activity were observed and ATP synthase, a major component of the respiratory chain, was identified as a calpain target. Interestingly we were able to protect the cells from these H(2)O(2)-induced effects and prevent calpain up-regulation with a natural antioxidant extracted from pine bark (Oligopin). These data strongly suggest that oxidative stress could impair satellite cell functionality via calpain-dependent pathways and that an antioxidant such as Oligopin could prevent apoptosis and calpain activation.

Topics: Antioxidants; Apoptosis; Calcium Signaling; Calpain; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytoplasm; Cytoplasmic Structures; Dipeptides; Flavonoids; Gene Expression; Humans; Hydrogen Peroxide; Mitochondrial Proton-Translocating ATPases; Myoblasts; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phenols; Pinus; Plant Extracts; Polyphenols; Protein Carbonylation; Satellite Cells, Skeletal Muscle; Up-Regulation

The islet in type 2 diabetes (T2DM) and the brain in neurodegenerative diseases share progressive cell dysfunction, increased apoptosis, and accumulation of locally expressed amyloidogenic proteins (islet amyloid polypeptide (IAPP) in T2DM). Excessive activation of the Ca(2+)-sensitive protease calpain-2 has been implicated as a mediator of oligomer-induced cell death and dysfunction in neurodegenerative diseases. To establish if human IAPP toxicity is mediated by a comparable mechanism, we overexpressed human IAPP in rat insulinoma cells and freshly isolated human islets. Pancreas was also obtained at autopsy from humans with T2DM and nondiabetic controls. We report that overexpression of human IAPP leads to the formation of toxic oligomers and increases beta cell apoptosis mediated by increased cytosolic Ca(2+) and hyperactivation of calpain-2. Cleavage of alpha-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca(2+)-calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM.

Topics: Adult; Aged; Aged, 80 and over; Amyloid; Animals; Apoptosis; Calcium; Calpain; Cell Line, Tumor; Cell Survival; Cytosol; Diabetes Mellitus, Type 2; Dipeptides; Enzyme Activation; Female; Humans; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Male; Middle Aged; Protein Transport; Rats; Recombinant Fusion Proteins; Spectrin

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

CEACAM1-4S (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I membrane protein with a short (12-amino acid) cytoplasmic tail. Wild type CEACAM1-4S-transfected MCF7 cells form glands with lumena when grown in 3D culture, while null mutations of two putative phosphorylation sites (T457A and S459A) in the cytoplasmic domain fail to undergo lumen formation. When gene chip analysis was performed on mRNA isolated from both wild type and T457A,S459A mutated CEACAM1-4S-transfected MCF7 cells grown in 3D culture, calpain-9 (CAPN9) was identified out of over 400 genes with a >2 log 2 difference as a potential inducer of lumen formation. Inhibition of CAPN9 expression in MCF7/CEACAM1-4S cells by RNAi or by calpeptin or PD150606 inhibited lumen formation. Transfection of CAPN9 into wild type MCF7 cells restores lumen formation demonstrating that calpain-9 may play a critical role in lumen formation. Additionally, we demonstrate that the apoptosis related kinase, PKC-delta, is activated by proteolytic cleavage during lumen formation exclusively in wild type CEACAM1-4S-transfected MCF7 cells grown in 3D culture and that lumen formation is inhibited by either RNAi to PKC-delta or by the PKC-delta inhibitor rottlerin.

Topics: Acrylates; Antigens, CD; Apoptosis; Blotting, Western; Breast Neoplasms; Calpain; Cell Adhesion Molecules; Cell Line, Transformed; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dipeptides; Epithelial Cells; Female; Gene Deletion; Gene Expression Regulation; Humans; Models, Biological; Mutation; Oligonucleotide Array Sequence Analysis; Protein Kinase C-delta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na(+)/Ca(2+) exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate-induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate-induced jump in cytosolic Ca(2+), completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate-treated hippocampal neurons, the initial increase in cytosolic Ca(2+) that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD.

Topics: Animals; Calcium; Calpain; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Membrane Potential, Mitochondrial; Mitochondria; Neurons; Neurotoxins; Rats; Sodium-Calcium Exchanger; Time Factors

Although cardiac hypertrophy initially ensues as a compensatory mechanism, it often culminates in congestive heart failure. Based on our earlier studies that calpain and beta3 integrin play cell death and survival roles, respectively, during pressure-overload (PO) hypertrophy, we investigated if the loss of beta3 integrin signaling is a potential mechanism for calpain-mediated cardiomyocyte death during PO. beta3 Integrin knockout (beta3) and wild-type mice were used to induce either moderate or severe PO in vivo for short-term (72-hour) and long-term (4-week) transverse aortic constriction. Whereas wild-type mice showed no changes during moderate PO at both time points, beta3 mice exhibited both enrichment of the mu-calpain isoform and programmed cell death of cardiomyocytes after 4-week PO. However, with severe PO that caused increased mortality in both mice groups, cell death was observed in wild-type mice also. To study calpain's role, calpeptin, a specific inhibitor of calpain, was administered through an osmotic mini-pump at 2.5 mg/kg per day beginning 3 days before moderate transverse aortic constriction or sham surgery. Calpeptin administration blocked both calpain enrichment and myocardial cell death in the 4-week PO beta3 mice. Because beta3 integrin contributes to cardioprotective signaling, these studies indicate that the loss of specific integrin function could be a key mechanism for calpain-mediated programmed cell death of cardiomyocytes in PO myocardium.

Topics: Animals; Apoptosis; Calpain; Cardiomegaly; Dipeptides; Heart Failure; Hypertrophy; Integrin beta3; Integrins; Mice; Mice, Knockout; Myocardium; Myocytes, Cardiac; Pressure; Signal Transduction

Exposure of procoagulant phosphatidylserine (PS) on the surface of activated platelets is not readily reversible and this may propagate thrombosis. Persistence of PS exposure may be attributed, at least in part, to a continued reduction of the activity of aminophospholipid translocase (APLT), that transports PS from the outer to the inner membrane leaflet. We investigated whether calpain is involved in the inhibition of APLT activity. In flow cytometric investigations, using the inhibitors calpeptin or E64d at a concentration that blocks calpain activation, we found that calpain is not responsible for the reduction in APLT activity that results in persistence of PS exposure. Unexpectedly, we found that the inhibitors had additional effects independent of blocking calpain. Incubation of resting platelets with calpeptin resulted in a subpopulation of platelets with increased intracellular Ca(2+) and persistent PS exposure. The inhibitors also increased the proportion of platelets with persistent PS exposure in suspensions stimulated with thrombin and/or collagen or the Ca(2+)-ionophore A23187 under conditions in which calpain was not activated or in which its activation was completely blocked; P-selectin expression on thrombin and/or collagen-stimulated platelets was inhibited. Furthermore, in stimulated platelets, calpeptin increased the proportion of the PS-exposing platelets expressing a second apoptotic hallmark, collapsed mitochondrial inner membrane potential (DeltaPsi(m)). These additional effects of calpeptin on platelet regulation of intracellular Ca(2+) levels and apoptotic-like events should be taken into account when it is used as an inhibitor of calpain.

Topics: Apoptosis; Blood Platelets; Calcimycin; Calcium Signaling; Calpain; Cell Separation; Cells, Cultured; Dipeptides; Enzyme Activation; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Phosphatidylserines; Phospholipid Transfer Proteins; Platelet Activation; Thrombin

Host cell death induced by Entamoeba histolytica is an important mechanism for both host defence and microbial immune evasion during human amoebiasis. However, the signalling pathways underlying cell death induced by E. histolytica are not fully understood. This study investigated the involvement of the protein tyrosine phosphatases (PTPs) SHP-1 and SHP-2 in the dephosphorylation associated with E. histolytica-induced host cell death. Incubation with E. histolytica resulted in a marked decrease in protein tyrosine phosphorylation levels and degradation of SHP-1 or SHP-2 in Jurkat cells. Pre-treatment of cells with a calpain inhibitor, calpeptin, impeded the amoeba-induced dephosporylation and cleavage of SHP-1 or SHP-2. Additionally, inhibition of PTPs with phenylarsine oxide (PAO) attenuated Entamoeba-induced dephosphorylation and DNA fragmentation in Jurkat T cells. These results suggest that calpain-dependent cleavage of SHP-1 and SHP-2 may contribute to protein tyrosine dephosphorylation in Jurkat T cell death induced by E. histolytica.

Topics: Arsenicals; Calpain; Cell Death; Dipeptides; Entamoeba histolytica; Enzyme Inhibitors; Humans; Jurkat Cells; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; T-Lymphocytes

Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose dependently with the calpain inhibitor calpeptin (50-250 microg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased, with down-regulation of proapoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of proapoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.

Topics: Animals; Axons; Blotting, Western; Calpain; Cell Death; Cysteine Proteinase Inhibitors; Dipeptides; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Fluorescent Antibody Technique; Gliosis; In Situ Nick-End Labeling; Inflammation; Male; Multiple Sclerosis; Nerve Tissue Proteins; Oligodendroglia; Rats; Rats, Inbred Lew; Spinal Cord; Tissue Embedding

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-kappaB through the polymerization-mediated depletion of I-kappaBalpha without IKK activation. This NF-kappaB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-kappaBalpha, which raises the question of how the polymerized I-kappaBalpha can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-kappaBalpha polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-kappaBalpha polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-kappaBalpha polymers in HEK-293 cells in case of TGase 2 transfection either with I-kappaBalpha or I-kappaBalpha mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-kappaBalpha polymers and I-kappaBalpha, concurrent with an inhibition of NF-kappaB activity in MDA-MB-231 cells. This suggests that mu-calpain proteasome-dependent I-kappaBalpha polymer degradation may contribute to cancer progression through constitutive NF-kappaB activation.

Topics: Calpain; Cell Line, Tumor; Cystamine; Dipeptides; GTP-Binding Proteins; Humans; I-kappa B Proteins; Neoplasms; NF-KappaB Inhibitor alpha; Proteasome Endopeptidase Complex; Protein Glutamine gamma Glutamyltransferase 2; RNA, Small Interfering; Transglutaminases; Ubiquitin

Idiopathic inflammatory myopathies (IIMs), comprising of polymyositis, dermatomyositis, and inclusion-body myositis, are characterized by muscle weakness and various types of inflammatory changes in muscle cells. They also show non-inflammatory changes, including perifascicular atrophy, mitochondrial changes, and amyloid protein accumulation. It is possible that some molecules/mechanisms bridge the extracellular inflammatory stimulation and intracellular non-inflammatory changes. One such mechanism, Ca(2+) influx leading to calpain activation has been proposed. In this study, we demonstrated that post-treatment with calpeptin (calpain inhibitor) attenuated intracellular changes to prevent apoptosis (Wright staining) through both mitochondrial pathway (increase in Bax:Bcl-2 ratio) and endoplasmic reticulum stress pathway (activation of caspase-12), which were induced by interferon-gamma (IFN-γ) stimulation in rat L6 myoblast cells. Our results also showed that calpeptin treatment inhibited the expression of calpain, aspartyl protease cathepsin D, and amyloid precursor protein. Thus, our results indicate that calpain inhibition plays a pivotal role in attenuating muscle cell damage from inflammatory stimulation due to IFN-γ, and this may suggest calpain as a possible therapeutic target in IIMs.

Topics: Analysis of Variance; Animals; Apoptosis; Blotting, Western; Calpain; Cell Line; Cells, Cultured; Dipeptides; Inflammation; Interferon-gamma; Myoblasts; Rats

Research on fertilization in mammalian species has revealed that Ca(2+) is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca(2+) is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca(2+) plays a pivotal role. Calpain-1 and calpain-2 are Ca(2+)-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca(2+). Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

Topics: Acrosome Reaction; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cytoskeleton; Dipeptides; Guinea Pigs; Leupeptins; Male; Microscopy, Electron; Spectrin; Sperm Capacitation; Spermatozoa

Pulmonary fibrosis is characterized by progressive worsening of pulmonary function leading to a high incidence of death. Currently, however, there has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on cytokines being associated with lung fibrosis, including interleukin (IL)-6 and transforming growth factor (TGF)-β1. We reported recently that two substances (ATRA and thalidomide) have preventive effects on pulmonary fibrosis by inhibiting IL-6-dependent proliferation and TGF-β1-dependent transdifferentiation of lung fibroblasts. Rheumatoid arthritis is a chronic autoimmune disorder, and its pathogenesis is also characterized by an association with several cytokines. It has been reported that calpain, a calcium-dependent intracellular cysteine protease, plays an important role in the progression of rheumatoid arthritis. In this study, we examined the preventive effect of Calpeptin, a calpain inhibitor, on bleomycin-induced pulmonary fibrosis. We performed histological examinations and quantitative measurements of IL-6, TGF-β1, collagen type Iα1 and angiopoietin-1 in bleomycin-treated mouse lung tissues with or without the administration of Calpeptin. Calpeptin histologically ameliorated bleomycin-induced pulmonary fibrosis in mice. Calpeptin decreased the expression of IL-6, TGF-β1, angiopoietin-1 and collagen type Iα1 mRNA in mouse lung tissues. In vitro studies disclosed that Calpeptin reduced (i) production of IL-6, TGF-β1, angiopoietin-1 and collagen synthesis from lung fibroblasts; and (ii) both IL-6-dependent proliferation and angiopoietin-1-dependent migration of the cells, which could be the mechanism underlying the preventive effect of Calpeptin on pulmonary fibrosis. These data suggest the clinical use of Calpeptin for the prevention of pulmonary fibrosis.

Topics: Angiopoietin-1; Animals; Bleomycin; Calpain; Cell Line, Transformed; Cell Movement; Cell Proliferation; Collagen Type I; Dipeptides; Female; Fibroblasts; Humans; Interleukin-6; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta1

Massive T cell infiltration into the central nervous system is a hallmark of multiple sclerosis (MS) and its rodent model experimental autoimmune encephalomyelitis (EAE), resulting in the induction of many of the pathophysiological events that lead to neuroinflammation and neurodegeneration. Thus, blocking T cell migration into the central nervous system may reduce disease severity in MS and EAE. One potential target for reducing T cell migration is inhibition of the Ca(2+)-activated neutral protease calpain. Previous studies in other cell types have demonstrated that migration is reduced by incubation of cells with calpain inhibitors. Thus, we hypothesize that calpain inhibition will reduce migration of T cells in response to and toward the chemokine CCL2. To test this hypothesis, the intracellular free Ca(2+) levels in Jurkat E6-1 T cells was first measured by the fura-2 assay to assess whether the intracellular ion environment would support calpain activation. The intracellular free Ca(2+) levels were found to increase in response to CCL2. The cells were next treated with the calpain inhibitor calpeptin in a multiwelled Boyden chamber with CCL2 used as the chemoattractant. These studies demonstrate that inhibition of calpain with its inhibitor calpeptin produces a dose-dependent inhibition of chemotaxis. Calpain activity, as measured by live cell imaging, was also increased in response to CCL2, providing further evidence of its involvement in the process of chemotaxis and migration. These studies provide evidence for the involvement of calpain in the mechanisms of chemotaxis and warrants further exploration in MS patient and EAE animal samples.

Topics: Calcium; Calpain; Chemokine CCL2; Chemotaxis, Leukocyte; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Fura-2; Glycoproteins; Humans; Jurkat Cells; Signal Transduction; T-Lymphocytes

Calpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO(*), specifically detected by electron spin resonance. This response was abolished by inhibition of calpain with N-acetyl-leucyl-leucyl-norleucinal (ALLN) or Calpeptin. Both also diminished membrane-specific calpain activation by VEGF, which was intriguingly attenuated by silencing ezrin with RNA interference. A rapid membrane colocalization of calpain and ezrin occurred as short as 10 minutes after VEGF stimulation. AKT, AMP-dependent kinase (AMPK), and endothelial nitric oxide synthase (eNOS)(s1179) phosphorylations in VEGF-stimulated endothelial cells were markedly enhanced, which were however significantly attenuated by either ALLN, Calpeptin, or ezrin small interfering RNA, as well as by Wortmannin or compound C (respectively for phosphatidylinositol 3-kinase [PI3K] or AMPK). The latter 3 also abolished VEGF induction of NO(*). These data indicate that AMPK and AKT are both downstream of PI3K and that AKT activation is partially dependent on AMPK. The interrelationship between AMPK and AKT, although known to be individually important in mediating VEGF activation of eNOS, is clearly characterized. Furthermore, AMPK/AKT/eNOS(s1179) was found downstream of a calpain/ezrin membrane interaction. These data no doubt provide new insights into the long mystified signaling gap between VEGF receptors and PI3K/AKT or AMPK-dependent eNOS activation. In view of the well-established significance of VEGF-dependent angiogenesis, these findings might have broad and important implications in cardiovascular pathophysiology.

Topics: AMP-Activated Protein Kinases; Animals; Aorta; Calpain; Cattle; Cells, Cultured; Cytoskeletal Proteins; Dipeptides; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Hydrogen Peroxide; Leupeptins; Membrane Proteins; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

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

Podoplanin/PA2.26 antigen is a small transmembrane mucin expressed in different types of cancer where it is associated with increased cell migration, invasiveness and metastasis. Little is known about the mechanisms that control podoplanin expression. Here, we show that podoplanin synthesis can be controlled at different levels. We analyzed podoplanin expression in a wide panel of tumour cell lines. The podoplanin gene (PDPN) is transcribed in cells derived from sarcomas, embryonal carcinomas, squamous cell carcinomas and endometrial tumours, while cell lines derived from colon, pancreatic, ovarian and ductal breast carcinomas do not express PDPN transcripts. PDPN is expressed as two mRNAs of approximately 2.7 and approximately 0.9 kb, both of which contain the coding sequence and arise by alternative polyadenylation. Strikingly, in most of the cell lines where PDPN transcripts were found, no podoplanin or only very low levels of the protein could be detected in Western blot. Treatment of several of these cell lines with the calpain inhibitor calpeptin resulted in podoplanin accumulation, whereas lactacystin, a specific inhibitor of the proteasome, had no effect. In vitro experiments showed that podoplanin is a substrate of calpain-1. These results indicate that at least in some tumour cells absence or reduced podoplanin protein levels are due to post-translational calpain-mediated proteolysis. We also report in this article the identification of a novel podoplanin isoform that originates by alternative splicing and differs from the standard form in lacking two cytoplasmic residues (YS). YS dipeptide is highly conserved across species, suggesting that it might be functionally relevant.

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; Calpain; Cell Line, Tumor; Dipeptides; Gene Expression; Glycoproteins; Humans; Membrane Glycoproteins; Molecular Sequence Data; Neoplasms; Protein Processing, Post-Translational; RNA, Messenger; Transcriptional Activation

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.

Topics: Alternative Splicing; Antineoplastic Agents; Apoptosis; Biopsy; Calpain; Cell Line, Tumor; Cell Nucleolus; Cisplatin; Cytoplasm; Dipeptides; Dysplastic Nevus Syndrome; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Muscle Proteins; Neoplasm Metastasis; Nevus; RNA, Messenger; Skin Neoplasms

Differentiation of erythroid cells is regulated by cell signaling pathways including those that change the intracellular concentration of calcium. Calcium-dependent proteases have been shown previously to process and regulate the activity of specific transcription factors. We show here that the protein levels of upstream stimulatory factor (USF) increase during differentiation of murine erythroleukemia (MEL) cells. USF was subject to degradation by the Ca(2+)-dependent protease m-calpain in undifferentiated but not in differentiated MEL cells. Treatment of MEL cells with the specific calpain inhibitor calpeptin increased the levels of USF and strongly induced expression of the adult alpha- and beta-globin genes. The induction of globin gene expression was associated with an increase in the association of USF and RNA po ly mer ase II with regulatory elements of the beta-globin gene locus. Calpeptin also induced high level alpha- and beta-globin gene expression in primary CD71-positive erythroid progenitor cells. The combined data suggest that inhibition of calpain activity is required for erythroid differentiation-associated increase in globin gene expression.

Topics: Animals; Calpain; Cell Differentiation; Dipeptides; Enzyme Inhibitors; Erythroid Cells; Female; Gene Expression Regulation; Globins; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred C57BL; Murinae; Promoter Regions, Genetic; RNA Polymerase II; Upstream Stimulatory Factors

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca(2+)-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 + or - 2% pre-MI to 30 + or - 4% with MI only vs. 41 + or - 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 + or - 2 microl pre-MI to 73 + or - 4 microl with MI only vs. 55 + or - 4 microl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

Topics: Animals; Apoptosis; Calpain; Cardiotonic Agents; Caspase 3; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Enzyme Activation; Injections, Intravenous; Injections, Subcutaneous; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocardial Infarction; Myocardium; Stroke Volume; Time Factors; Ultrasonography; Ventricular Function, Left; Ventricular Remodeling

Gossypol, a cottonseed extract derivative, acts as a BH3-mimetic, binding to the BH3 pocket of antiapoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge on the molecular underpinnings of its downstream effects is limited. Since chronic lymphocytic leukemia (CLL) cells express high levels of antiapoptotic proteins that act as a survival mechanism for these replicationally quiescent lymphocytes, we investigated whether gossypol induces apoptosis in these cells and what mechanism underlies gossypol-mediated cytotoxicity. Gossypol induced cell death in a concentration- and time-dependent manner; 24-hour incubation with 30 microM gossypol resulted in 50% cell death (median; range, 10%-80%; n = 47) that was not abrogated by pan-specific caspase inhibitor. Starting at 4 hours, the mitochondrial outer membrane was significantly permeabilized (median, 77%; range, 54%-93%; n = 15). Mitochondrial outer membrane permeabiliztaion (MOMP) was concurrent with increased production of reactive oxygen species (ROS); however, antioxidants did not abrogate gossypol-induced cell death. Mitochondrial membrane permeabilization was also associated with loss of intracellular adenosine triphosphate (ATP), activation of BAX, and release of cytochrome c and apoptosis-inducing factor (AIF), which was translocated to the nucleus. Blocking AIF translocation resulted in a decreased apoptosis, suggesting that AIF contributes to gossypol-mediated cytotoxicity in CLL lymphocytes.

Topics: Acetylcysteine; Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Biological Transport, Active; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dipeptides; Gossypol; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondrial Membranes; Molecular Mimicry; Reactive Oxygen Species; Superoxides

Calpains are a family of calcium-dependent proteases with abundant expression in the CNS, and potent in cleaving some synaptic components. Assessment of calpain activity by its fluorescent substrate, Boc-Leu-Met-CMAC, revealed that cultured neurons display a significant level of constitutive enzyme activity. Notably, calpain activity differs in distinct neuronal populations, with a significantly higher level of activity in GABAergic cells. Using selectively-enriched cultures of fast-spiking GABAergic interneurons, we show that calpain activity partially contributes to the post-translational down regulation of SNAP-25, a calpain substrate, in differentiated GABA cells. In addition, we demonstrate that SNAP-25 is cleaved by calpain in response to acute seizures induced by intraperitoneal kainate injection in vivo. These data indicate that calpains in neurons are active even at physiological calcium concentrations and that different levels of calpain activation in selected neuron subtypes may contribute to the pattern of synaptic protein expression.

Topics: Animals; Brain-Derived Neurotrophic Factor; Calcium; Calpain; Cells, Cultured; Coumarins; Dipeptides; Embryo, Mammalian; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; Kainic Acid; Mice; Mice, Inbred C57BL; Neurons; Patch-Clamp Techniques; Protein Isoforms; Rats; Seizures; Synaptosomal-Associated Protein 25; Synaptosomes

It is widely known that the pathophysiology of idiopathic Parkinson's disease (PD) is associated with neurodegeneration and inflammatory responses in the midbrain substantia nigra. However, the possibility of neurodegeneration and inflammatory responses in other areas of the central nervous system (CNS) in course of the pathogenesis of PD remains to be explored. In this investigation, we provide evidence in support of the hypothesis that spinal cord, the final coordinator of movement, is also involved during parkinsonian degeneration using two distinct experimental parkinsonism models induced by the neurotoxin 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) and the environmental toxin rotenone. A key focus of our study is the role that calpain, a Ca(2+)-activated neutral protease, plays in disrupting the structural-functional integrity of the spinal cord in the context of spinal cord degeneration in experimental parkinsonism. We examined the mechanisms of calpain-mediated neuronal death in differentiated spinal cord motoneuron cultures following exposure to the active parkinsonian toxins 1-methyl-4-phenyl-pyridinium ion (MPP(+)) and rotenone and also tested the neuroprotective efficacy of calpeptin, a calpain inhibitor, in these cell culture models of experimental parkinsonism. Our results implied that spinal cord motoneurons could be a potential extranigral target of neurodegeneration during pathogenesis of PD in the CNS and that calpain inhibition could provide neuroprotection.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Apoptosis; Calpain; Cell Line; Dipeptides; Humans; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neuroprotective Agents; Neurotoxins; Parkinson Disease; Parkinsonian Disorders; Rotenone; Spinal Cord; Substantia Nigra; Uncoupling Agents

Calpeptin inhibits myoblast fusion by inhibiting the activity of calpain. However, the mechanism by which calpeptin inhibits myogenesis is not completely understood. This study examined how calpeptin affects the expression of the myogenic regulatory factors (MRFs) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in differentiating C2C12 myoblasts. Consistent with previous reports, calpeptin inhibited the induction of mu-calpain and the formation of myotubes in these cells. In particular, calpeptin inhibited the expression of the early and mid differentiation markers including MyoD, Myf5, myogenin, and MRF4 as well as the expression of the late markers such as troponin T and myosin heavy chain (MyHC). Calpeptin also suppressed the phosphorylation of p38 MAPK in C2C12 cells. SB203580, a specific p38 inhibitor, prevented the expression of the muscle-specific markers and their fusion into myotubes in these cells, which was further accelerated in the presence of calpeptin. These findings suggest that calpeptin inhibits the myogenesis of skeletal muscle cells by down-regulating the MRFs and involving p38 MAPK signaling.

Topics: Animals; Calpain; Cell Differentiation; Cell Fusion; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Dipeptides; MAP Kinase Signaling System; Mice; Muscle Development; Muscle Fibers, Skeletal; Myoblasts; MyoD Protein; Myogenic Regulatory Factors; Myosin Heavy Chains; p38 Mitogen-Activated Protein Kinases; Troponin T

Brain-pancreas relative protein (BPRP) is a novel protein that we found in our laboratory. Previously we demonstrated that it is involved in ischemia and depression. In light of the putative association between diabetes and clinical depression, and the selective expression of BPRP in brain and pancreas, the present study examined whether BPRP levels are affected by induction of diabetes by alloxan injection in rats and exposure to high glucose levels in PC12 cells. Western blot and immunohistochemical analyses revealed that BPRP levels were decreased in the hippocampal CA1 neurons of diabetic rats 4 and 8 weeks post-alloxan injection and in PC12 cells 48 h after exposure to high concentrations of glucose. BPRP protein levels were not affected by osmolarity control treatments with mannitol. Follow-up pharmacological experiments in PC12 cells revealed that glucose-induced BPRP down-regulation was markedly attenuated by the calpain inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) or calpeptin, but not the proteasome-specific inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132). The ability of calpain inhibitors to specifically counter the effects of high glucose exposure on BPRP levels further suggests that BPRP and calpain activity may contribute to diabetes complications in the central nervous system.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Experimental; Dipeptides; Down-Regulation; Glucose; Half-Life; Hippocampus; Insulin; Leupeptins; Male; Nerve Tissue Proteins; Neurons; PC12 Cells; Rats; Rats, Sprague-Dawley; Time Factors

In recent studies we have demonstrated that arsenic (As) metabolites change the composition of neuronal cytoskeletal proteins in vivo and in vitro. To further examine the mechanism of arsenic-induced neurotoxicity with various arsenate metabolites (iAsV, MMAV and DMAV) and arsenite metabolites (iAsIII, MMAIII and DMAIII), we investigated the role of the proteolytic enzyme calpain and its involvement in the cleavage of p35 protein to p25, and also mRNA expression levels of calpain, cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (gsk3ss). A HeLa cell line transfected with a p35 construct (HeLa-p35) was used as a model, since all other proteins such as calpain, CDK5 and GSK3beta are already present in HeLa cells as they are in neuronal cells. HeLa-p35 cells were incubated with various As metabolites and concentrations of 0, 10 and 30 microM for duration of 4 h. Subsequently the cells were either lysed to study their relative quantification levels of these genes or to be examined on their p35-protein expression. P35-RNA expression levels were significantly (p<0.01) increased by arsenite metabolites, while p35 protein was cleaved to p25 (and p10) after incubation with these metabolites. The cleavage of p35 is caused by calcium (Ca2+) induced activation of calpain. Inhibition of calpain activity by calpeptin prevents cleavage of p35 to p25. These results suggest that cleavage of p35 to p25 by calpain, probably As-induced Ca2+-influx, may explain the mechanism by which arsenic induces its neurotoxic effects.

Topics: Arsenic; Blotting, Western; Calcium; Calpain; Cyclin-Dependent Kinase 5; Cysteine Proteinase Inhibitors; Dipeptides; DNA Primers; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; HeLa Cells; Humans; Nerve Tissue Proteins; Neurotoxicity Syndromes; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.

Topics: Amino Acid Motifs; Amino Acid Sequence; Antineoplastic Agents; Apoptosis; Calpain; Cisplatin; Dipeptides; DNA Glycosylases; Enzyme Inhibitors; Guanine; HeLa Cells; Humans; Hydrogen Peroxide; Molecular Sequence Data; Oxidative Stress; Proteolysis

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Cardiomegaly; Caspase 3; Caspase Inhibitors; Cats; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Enzyme Activation; Gelsolin; Heart Failure; Histones; In Situ Nick-End Labeling; Injections, Intravenous; Ligation; Male; Myocytes, Cardiac; Phosphorylation; Pulmonary Artery; Time Factors

Alleles of IL1A-889(C>T) and IL1A+4845(G>T) are in linkage disequilibrium. Interleukin 1alpha (IL-1alpha) is produced as a precursor protein and cleaved at positions 117-118 by calpain, generating a mature protein for export. IL1A+4845 affects amino acids expressed at position 114 and hence may modulate calpain-mediated cleavage. We sought evidence for this mechanism in intact cells. Blood leukocytes from heterozygous donors released more IL-1alpha protein than cells from IL1A(1,1) donors, while release from IL1A(2,2) cells was variable. Genotype did not affect levels of IL-1alpha mRNA, so differential cleavage of the precursor is a feasible mechanism. However, genotype also had no effect on inhibition of IL-1alpha release by pretreatment with calpain inhibitors, and calpain inhibitors reduced IL-1alpha and tumor necrosis factor alpha mRNA levels. Hence, calpain inhibitors probably affect inhibition of signal transduction pathway rather than cleavage of IL-1alpha protein. As ratios of mu-calpain/calpastatin were lowest in heterozygous donors, genetically determined IL-1alpha levels may modulate transcription of calpain and calpastatin. This could reduce the impact of IL1A genotype on IL-1alpha secretion and amplify individual variation in levels generated in culture.

Topics: Acrylates; Calpain; Cells, Cultured; Dipeptides; Glycoproteins; Humans; Interleukin-1alpha; Leupeptins; Polymorphism, Genetic; RNA, Messenger

Activation of calpains by calcium flux leading to talin cleavage is thought to be an important process of LFA-1 activation by inside-out signalling. Here, we tested the effects of the calcium ionophore ionomycin and calpain inhibitor calpeptin on LFA-1-mediated adhesion of a T cell hybridoma line, cytotoxic T cells and primary resting T cells. Ionomycin activated LFA-1-mediated adhesion of all three types of T cells, and calpeptin inhibited the effects of ionomycin. However, calpeptin also inhibited activation of LFA-1 by PMA, which did not induce calcium flux. Cleavage of talin was undetectable in ionomycin-treated T cells. Furthermore, treatment with ionomycin and calpeptin induced apoptosis of T cells. Inhibitors of phosphatidyl Inositol-3 kinase inhibited activation of LFA-1 by ionomycin, but not by PMA, whereas the protein kinase C inhibitor inhibited the effects of PMA, but not ionomycin. Thus, activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage.

Topics: Acrylates; Androstadienes; Animals; Apoptosis; Calpain; Cell Adhesion; Cell Line, Tumor; Chromones; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Hybridomas; Ionomycin; Ionophores; Lymphocyte Function-Associated Antigen-1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Receptors, Antigen, T-Cell; T-Lymphocytes; Talin; Wortmannin

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting human platelets by extruding Ca(2+) from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca(2+) efflux in platelets, it is a key point for regulation of platelet Ca(2+) metabolism. PMCA has been shown to be an excellent substrate for the Ca(2+)-activated cysteine protease calpain, a major platelet protein that is turned on during platelet activation. The objectives of the present work were to determine if PMCA is degraded during thrombin- and collagen-mediated platelet activation, and if calpain is responsible. The kinetics of PMCA degradation during platelet activation were analysed using SDS polyacrylamide gel electrophoresis and immunoblotting. The role of calpain was tested using the calpain inhibitors calpeptin and ALLN. Platelet activation mediated by both collagen and thrombin resulted in degradation of 60% of platelet PMCA within 18 minutes. Calpeptin and ALLN significantly inhibited the rate and extent of PMCA degradation. We conclude that calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca(2+) regulation and, therefore, to platelet function.

Topics: Blood Platelets; Calcium; Calcium-Transporting ATPases; Calpain; Cell Membrane; Collagen; Dipeptides; Enzyme Activation; Humans; Leupeptins; Platelet Activation; Thrombin

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Dipeptides; DNA Cleavage; Entamoeba histolytica; Entamoebiasis; Enzyme Activation; Host-Parasite Interactions; Humans; Jurkat Cells; Parasitology; T-Lymphocytes

Evidence has implicated apoptosis as a mechanism underlying cell death in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous agents such as TNF-alpha, INF-gamma, IL-1beta and others stress signals activate the sphingomyelin pathway increasing ceramide levels. Ceramide triggers apoptotic pathways while inhibiting survival signalling, and is involved in the regulation of intracellular Ca(2+) homeostasis and compartmentalisation. The contribution of caspases in neuronal apoptosis has been highlighted by the increased survival exerted by caspase inhibition, but the involvement of calpains during neuronal apoptosis and the potential benefit of their inhibition is still controversial. In the present paper, we have analysed the contribution of caspases and calpains to cell death of CAD cells, a catecholaminergic cell line of mesencephalic origin, following C2-ceramide exposure. Ceramide caused CAD cell death by a dose and time dependant mechanism. 25microM of C2-ceramide caused apoptosis. Analysis of activation of caspases and calpains by differential cleavage of alpha-fodrin showed that although calpains are activated before caspases following C2-ceramide exposure, only caspase inhibition increased cell survival. These results demonstrate the activation of caspases and calpains in C2-ceramide-induced cell death, and support the role of caspase inhibition as a neuroprotective strategy and a plausible therapeutic approach to decrease catecholaminergic cell death.

Topics: Analysis of Variance; Animals; Calpain; Caspases; Cell Death; Cell Line; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Mice; Neurons; Neuroprotective Agents; Sphingosine; Tetrazolium Salts; Thiazoles; Time Factors

Proteolytic activity in sheared human umbilical vein endothelial cells (HUVECs) was measured using a fluorogenic substrate and laser scanning confocal microscopy to clarify the key role of an intracellular Ca(2+)-sensitive protease, calpain, in these cells in response to shear stress. Within physiological shear range, activity in the cells was enhanced in shear-dependent fashion. Short interfering RNA-induced silencing of m-calpain, but not of micro-calpain, suppressed the activity. Either removal of extracellular Ca(2+) or application of an intracellular Ca(2+) chelator (BAPTA/AM) or nonselective cation channel blocker (Gd(3+)) reduced proteolytic activity. Furthermore, activity was suppressed by phosphatidylinositol bisphosphate (PIP(2)) chelator (neomycin) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002); in contrast, activity, which was partially inhibited by ERK kinase inhibitor (U0126, PD98059), was unaffected by PLC inhibitor (U73122). Moreover, Akt phosphorylation downstream of PI3K, which was elicited by shear, was attenuated by neomycin but not by calpain inhibitor (calpeptin). Following assessment of shear stress-induced focal adhesion (FA) and cytoskeletal dynamics using interference reflection/green fluorescence protein-actin microscopy, we found that either calpain or PI3K inhibition impaired shear stress-induced polarization of FAs via stabilization of FA structures. Additionally, HUVEC alignment and cytoskeletal remodeling, which was accompanied by calpain-mediated cleavage of vinculin and talin, were also elicited by prolonged application of shear and impaired by m-calpain knockdown. Thus, these results revealed that physiological shear stress elicits Ca(2+) influx-sensitive activation of m-calpain in HUVECs. This activity is facilitated primarily through the PI3K pathway; furthermore, it is essential for subsequent FA reorganization and cell alignment under shear conditions.

Topics: Actins; Calcium; Calpain; Cell Polarity; Cells, Cultured; Chromones; Cytoskeleton; Dipeptides; Endothelial Cells; Flavonoids; Focal Adhesions; Gadolinium; Humans; Microscopy, Confocal; Morpholines; Neomycin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pseudopodia; RNA, Small Interfering; Stress, Mechanical; Talin; Vinculin

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.

Topics: Calcium-Binding Proteins; Calpain; Cell Line; Cytoskeletal Proteins; Dipeptides; Egtazic Acid; Endopeptidase K; Eye Proteins; Glaucoma; Glycoproteins; Humans; Ionomycin; Mutation, Missense; Protein Conformation; Protein Structure, Tertiary; Transfection

Multiple sclerosis (MS) is a devastating autoimmune demyelinating disease of the central nervous system (CNS). This study investigated whether expression and activity of the calcium-activated protease calpain correlated with Th1/Th2 dysregulation in MS patients during states of relapse and remission. Calpain expression and activity were significantly increased in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to controls, with the highest expression and activity noted during relapse. Th1 cytokines were highest and Th2 cytokines were lowest in MS patients during relapse. Treatment with calpain inhibitor, calpeptin, decreased Th1 cytokines in PBMCs from MS patients. Calpain inhibitor also reduced degradation of myelin basic protein (MBP) by inhibiting the calpain secreted from MBP-specific T cells. Taken together, these results suggested calpain involvement in Th1/Th2 dysregulation in MS patients.

Topics: Biomarkers; Calcium; Calcium Signaling; Calpain; Cytokines; Dipeptides; Enzyme Inhibitors; Female; Humans; Male; Multiple Sclerosis; Myelin Basic Protein; Neutrophils; Recurrence; Th1 Cells; Th2 Cells; Up-Regulation

Prostate carcinoma is the most commonly diagnosed cancer in men and the second leading cause of death due to cancer in Western civilization. Androgen ablation therapy is effective in treating androgen-dependent tumors, but eventually, androgen-independent tumors recur and are refractory to conventional chemotherapeutics. Hence, the emergence of androgen independence is the most challenging problem in managing prostate tumors. We report a novel mechanism of androgen independence: calpain cleaves the androgen receptor (AR) into an androgen-independent isoform. In vitro and in vivo analyses show that calpain removes the COOH-terminal ligand binding domain generating a constitutively active molecule. Analysis of human prostate tumors indicates that several tumors express higher levels of this truncated AR than noncancerous prostate tissue. In transient transfection studies, the truncated AR is three to five times more potent than the full-length receptor in transactivating transcription. The androgen-independent Rv1 cells express high levels of the truncated AR, and treatment of these cells with a calpain inhibitor reduces truncated AR expression. In the absence of androgen, inhibition of calpain activity induces apoptosis. The HIV protease inhibitor amprenavir inhibits calpain activity and is also effective in inducing apoptosis in the Rv1 cell line. The cell culture studies were reproduced in a mouse xenograft model, where, in the absence of androgens, amprenavir significantly reduces tumor growth. Together, these studies indicate that calpain-dependent proteolysis of the AR may be a mechanism of androgen independence. The calpain inhibition studies suggest that inhibiting this activity may be a potential treatment for some androgen-independent prostate tumors.

Topics: Androgens; Animals; Apoptosis; Calpain; Carbamates; Cell Line, Tumor; Dipeptides; Enzyme Activation; Furans; Humans; Ionomycin; Male; Mice; Mice, Nude; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptors, Androgen; Sulfonamides; Transcriptional Activation; Transfection; Transplantation, Heterologous

The purpose of this study was to examine the importance of calpains in retinal ganglion cell (RGC) apoptosis and the protection afforded by calpain inhibitors against cell death.. Two different models of RGC apoptosis were used, namely the RGC-5 cell line after either intracellular calcium influx or serum withdrawal and retinal explant culture involving optic nerve axotomy. Flow cytometry analysis with Annexin V/PI staining was used to identify RGC-5 cells undergoing apoptosis after treatment. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis in retinal explant sections under various conditions. Serial sectioning was used to isolate the cell population of the ganglion cell layer (GCL). Western blotting was used to demonstrate calpain cleavage and activity by detecting cleaved substrates.. In the RGC-5 cell line, the authors reported the activation of mu-calpain and m-calpain after serum starvation and calcium ionophore treatment, with concurrent cleavage of known calpain substrates. They found that the inhibition of calpains leads to the protection of cells from apoptosis. In the second model, after a serial sectioning method to isolate the cells of the ganglion cell layer (GCL) on a retinal explant paradigm, protein analysis indicated the activation of calpains after axotomy, with concomitant cleavage of calpain substrates. The authors found that inhibition of calpains significantly protected cells in the GCL from cell death.. These results suggest that calpains are crucial for apoptosis in RGCs after calcium influx, serum starvation, and optic nerve injury.

Topics: Animals; Annexin A5; Apoptosis; Axotomy; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Flow Cytometry; Glaucoma; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Optic Nerve; Organ Culture Techniques; Propidium; Retina; Retinal Ganglion Cells

The extracellular matrix (ECM) of the atherosclerotic lesion is a crucial determinant of its stability, while its degradation by matrix metalloproteinases (MMPs) has been implied in plaque rupture. As accumulation of both MMP-derived collagen fragments and apoptotic smooth muscle cells (SMC) is observed at sites of plaque rupture, we tested the effect of polymerized and degraded type I collagen on the susceptibility of SMC to apoptosis.. Human SMC were cultured on monomeric or polymerized collagen, and collagen gels were degraded by collagenase. Apoptosis was evaluated using antibodies to active caspases and their substrates. Calpain and caspase activity were measured using fluorogenic substrates.. Culture of SMC on polymerized collagen led to increased apoptosis compared to culture on monomeric collagen. In addition, we observed a distinct proteolytic degradation of the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis (xIAP). As MMP-1 was strongly activated in SMC on polymerized collagen, we examined the effect of degraded collagen fragments on xIAP cleavage and apoptosis. Degraded collagen induced rapid proteolytic processing of xIAP identical to that on polymerized collagen. We identified calpains as the proteolytic enzymes responsible for xIAP processing as: i) they were rapidly activated by degraded collagen; ii) recombinant calpain II processed xIAP in an identical manner, and iii) inhibition of calpains by BAPTA or calpeptin abrogated xIAP degradation in intact cells. The functional consequence of xIAP processing by calpains was a loss of its caspase-inhibitory potential. Calpain activation distinctly preceded caspase activation, and inhibition of calpains suppressed apoptosis.. Collagen fragments proteolytically released from the ECM by MMPs may propagate apoptosis of SMC by calpain-mediated inactivation of anti-apoptotic proteins such as xIAP. This may be a novel mechanism of SMC apoptosis in biological settings of enhanced collagen degradation such as vascular remodeling, neointima formation, and atherosclerotic plaque rupture.

Topics: Apoptosis; Atherosclerosis; Calpain; Caspases; Cells, Cultured; Collagen; Collagenases; Dipeptides; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix; Gels; Humans; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Polymers; Recombinant Proteins; X-Linked Inhibitor of Apoptosis Protein

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dexamethasone; Dipeptides; Gene Expression; Glycoproteins; Hydrogen Peroxide; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myoblasts, Skeletal; Pepstatins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Sepsis; SKP Cullin F-Box Protein Ligases; Transfection; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Members of the C/EBP transcription factor family regulate cell differentiation and multiple other cellular functions. The cellular levels of C/EBPalpha, gamma, delta, epsilon, and Gadd153/CHOP are regulated in part by proteasome-dependent degradation. In contrast, mechanisms regulating the degradation of C/EBPbeta are poorly understood. We tested the hypothesis that the degradation of C/EBPbeta is calpain-dependent. Studies were performed in cultured L6 myotubes (a rat skeletal muscle cell line) because we have found previously that C/EBPbeta may be involved in the regulation of muscle proteolysis. Treatment of cultured L6 myotubes with the calpain inhibitors calpeptin and Calpain Inhibitor I and II resulted in increased C/EBPbeta concentrations but did not influence cellular levels of the other C/EBP transcription factor family members. Transfection of myoblasts with a plasmid expressing the endogenous calpain inhibitor calpastatin resulted in increased cellular levels of C/EBPbeta whereas the opposite result was observed in myoblasts overexpressing micro- or m-calpain. Co-immunoprecipitation provided evidence for protein-protein interaction between C/EBPbeta and micro- and m-calpain suggesting that C/EBPbeta may be a calpain substrate. This notion was supported by experiments in which immunoprecipitated C/EBPbeta was incubated with purified micro-calpain in a cell-free system. The increase in C/EBPbeta levels caused by inhibition of calpain activity was accompanied by increased C/EBPbeta DNA-binding and gene activation. The present results suggest that C/EBPbeta is degraded by a calpain-dependent mechanism in skeletal muscle cells and that the role of calpains is specific for C/EBPbeta among different members of the C/EBP transcription factor family.

Topics: Animals; Calpain; CCAAT-Enhancer-Binding Protein-beta; Cell Culture Techniques; Cell Line; Dipeptides; Dose-Response Relationship, Drug; Glycoproteins; Mice; Muscle Fibers, Skeletal; Muscle, Skeletal; Protein Isoforms; Rats

Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative stress.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Coronary Vessels; Cytosol; Dipeptides; Endothelium, Vascular; Flavonoids; Gene Expression Regulation, Enzymologic; Homocysteine; Hyperhomocysteinemia; Matrix Metalloproteinase 9; Mitochondria; Mitogen-Activated Protein Kinase 3; Oxidative Stress; Rats; Rats, Wistar; Uncoupling Agents

3-Chlorotyrosine, a bio-marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimer's disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic-necrotic continuum of concentration-dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 microm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa alpha-fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of alpha-fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl-induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Calcium; Calcium Channels; Calpain; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Hypochlorous Acid; Lysosomes; Mice; Microscopy, Electron, Transmission; Neocortex; Neurons; Nifedipine; Oxidants; Tetrazolium Salts; Thiazoles; Time Factors

Previous research in our laboratory has already shown the importance of the role played by ubiquitous calpains during myoblast migration. The aim of this study was to investigate calpain expression during myoblast migration and, to enhance this phenomenon via calpain stimulation. Ubiquitous calpains are members of a large family of calcium-dependent cysteine proteases. They play an important role in numerous biological and pathological phenomena, such as signal transduction, apoptosis, cell-cycle regulation, cell spreading, adhesion, invasion, myogenesis, and motility. Myoblast migration is a crucial step in myogenesis, as it is necessary for myoblast alignment and fusion to form myotubes. This study started by examining changes in calpain expression during migration, then investigated the possibility of activating myoblast migration via the stimulation of calpain expression and/or activity. The migration rate of myoblasts overexpressing mu- or milli-calpain was quantified. The results showed that calpain overexpression dramatically inhibited myoblast migration. Growth-factor treatments were then used to enhance myoblast migration. The results showed that treatment with IGF-1, TGF-beta1, or insulin induced a major increase in migration and caused a significant increase in m-calpain expression and activity. The increase in migration was totally inhibited by adding calpeptin, a calpain-specific inhibitor. These findings suggest that milli-calpain is involved in growth factor-mediated migration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caseins; Cell Movement; Dipeptides; Gene Expression; Growth Substances; Insulin; Mice; Myoblasts; Stress Fibers

Parkinson's disease (PD) is a movement disorder characterized by progressive degeneration of primarily the dopaminergic neurons in the substantia nigra (SN). The present study briefly describes our findings to support the hypothesis that there is a possibility of degeneration of spinal cord (SC) motoneurons in course of parkinsonism. In cell culture models of experimental parkinsonism, we examined the degeneration of ventral SC motoneuron cell line (VSC4.1) following exposure to two different toxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone. Our studies suggested calpain activation in the apoptosis of VSC4.1 motoneurons due to exposure to these parkinsonian toxins. Furthermore, our study showed the toxic effects of the dopaminergic toxin methamphetamine (METH) on VSC4.1 cells. The results strongly implicated a possible role for calpain in the mechanism of motoneuron apoptosis during parkinsonian degeneration, at large. Hence, we examined the neuroprotective efficacy of calpeptin, a specific inhibitor of calpain, in cell culture model of experimental parkinsonism.

Topics: Animals; Apoptosis; Calpain; Cell Culture Techniques; Dipeptides; Disease Models, Animal; DNA Fragmentation; Humans; In Situ Nick-End Labeling; Motor Neurons; Parkinsonian Disorders; Rats

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.

Topics: Acetamides; Acetophenones; Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Benzopyrans; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Histones; Humans; Indomethacin; Kinetics; Phenanthridines; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Pyrans; Spiro Compounds; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.

Topics: Animals; Bacterial Infections; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Oligopeptides; Rats; Rats, Sprague-Dawley; RNA, Messenger

RCP (Rab coupling protein) belongs to the recently identified Rab11-FIPs (Rab11 family of interacting proteins). All the Rab-FIP members have the ability to bind Rab11 tightly via a Rab-binding domain located near their C-termini. RCP belongs to the class I Rab11-FIP subfamily, characterized by the presence of a conserved C2 domain near its N-terminus. The function of this protein in Rab11-dependent membrane trafficking remains to be fully understood. In the present study, we have identified three putative PEST (Pro, Glu, Ser/Thr-rich) sequences in RCP. PEST motifs play a role in targeting a protein for proteolytic degradation. We have demonstrated that RCP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. Using a mutant, lacking the three PEST sequences, RCP(DeltaPEST), we demonstrated that they are necessary for the cleavage of RCP by calpains. When expressed in A431 cells, RCP(DeltaPEST) displays significantly greater localization to the plasma membrane, compared with the wild-type protein. Similarly, treatment with the calpain inhibitor, calpeptin, results in the redistribution of endogenous RCP to the periphery of the cell. We propose that once the Rab11/RCP-regulated cargo has been delivered from the endocytic recycling compartment to the plasma membrane, RCP is inactivated by calpain-mediated proteolysis.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Motifs; Amino Acid Sequence; Animals; Calcium; Calpain; Carrier Proteins; Cell Line, Tumor; Cell Membrane; Conserved Sequence; Dipeptides; Humans; Membrane Proteins; Mice; Secretory Vesicles; Sequence Alignment; Sequence Homology, Amino Acid

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.

Topics: Animals; Calcium; Calpain; Cell Movement; Cell Proliferation; Cell Size; Cells, Cultured; Dipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Matrix; Insulin; Insulin Secretion; Intracellular Fluid; Islets of Langerhans; Male; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction

There is some evidence suggesting that Ca2+ is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+ takes part in its regulation. However, the precise roles of Ca2+ in spermatogenesis remain to be elucidated. Calpains are a family of Ca(2+)-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca(2+)-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain's ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.

Topics: Acrosome Reaction; Animals; Blotting, Western; Calpain; Dipeptides; Dose-Response Relationship, Drug; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Confocal; Reverse Transcriptase Polymerase Chain Reaction; RNA; Spermatocytes; Spermatogenesis; Spermatozoa

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.

Topics: Actins; Aged; Bone and Bones; Bone Resorption; Calpain; Cells, Cultured; Dipeptides; Humans; Leucine; Osteoclasts; Recombinant Fusion Proteins; Talin

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 3; Caspases; Cell Fusion; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; Glutamic Acid; Humans; Ionomycin; Ionophores; Mice; Mitochondria; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Patch-Clamp Techniques; Proto-Oncogene Proteins c-bcl-2; Rats; Spectrin; Spinal Cord

In primary cultures of rat hepatocytes, exposure to arsenite causes a major decrease in dexamethasone (DEX)-mediated induction of CYP3A23 hemoprotein, with a minor decrease in CYP3A23 mRNA. Here we show that addition of heme did not prevent the arsenite-mediated decreases in CYP3A23 protein, and arsenite did not decrease intracellular glutathione levels, indicating that heme and glutathione were not limiting for formation of holoCYP3A23. We also investigated whether arsenite decreases CYP3A23 protein by increasing CYP3A23 degradation by the calpain pathway. The calpain inhibitor, calpeptin, caused greater than a 90% inhibition of calpain-mediated proteolysis, but had no effect on DEX-mediated induction of CYP3A23 protein following 24h treatments. However, calpeptin enhanced the effect of arsenite to decrease induction of CYP3A23 protein. In addition, in short-term studies, calpeptin appeared to be a suicidal inhibitor of CYP3A-catalyzed enzyme activity. Our findings suggest that CYP3A23 protein is not degraded by calpain-mediated proteolysis, even in the presence of arsenite.

Topics: Animals; Arsenites; Aryl Hydrocarbon Hydroxylases; Calpain; Cells, Cultured; Cytochrome P-450 CYP3A; Dipeptides; Dose-Response Relationship, Drug; Glutathione; Heme; Hepatocytes; Male; Rats; Rats, Inbred F344; Signal Transduction

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.

Topics: Age Factors; Animals; Animals, Newborn; Antibodies; Blotting, Western; Calcium; Calpain; Cells, Cultured; Cerebellum; Chromatography, High Pressure Liquid; Dipeptides; Dose-Response Relationship, Drug; Down-Regulation; Drug Interactions; Excitatory Amino Acid Agonists; Glutamic Acid; Glycoproteins; Ionomycin; Ionophores; Membrane Proteins; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Potassium; Rats; Rats, Wistar; Synaptosomal-Associated Protein 25; Time Factors

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

The cell body of a lesioned neuron must receive accurate and timely information on the site and extent of axonal damage, in order to mount an appropriate response. Specific mechanisms must therefore exist to transmit such information along the length of the axon from the lesion site to the cell body. Three distinct types of signals have been postulated to underlie this process, starting with injury-induced discharge of axon potentials, and continuing with two distinct types of retrogradely transported macromolecular signals. The latter include, on the one hand, an interruption of the normal supply of retrogradely transported trophic factors from the target; and on the other hand activated proteins emanating from the injury site. These activated proteins are termed "positive injury signals", and are thought to be endogenous axoplasmic proteins that undergo post-translational modifications at the lesion site upon axotomy, which then target them to the retrograde transport system for trafficking to the cell body. Here, we summarize the work to date supporting the positive retrograde injury signal hypothesis, and provide some new and emerging proteomic data on the system. We propose that the retrograde positive injury signals form part of a complex that is assembled by a combination of different processes, including post-translational modifications such as phosphorylation, regulated and transient proteolysis, and local axonal protein synthesis.

Topics: Animals; Axons; Calpain; Dipeptides; DNA-Binding Proteins; Electrophoresis, Gel, Two-Dimensional; Genes, Tumor Suppressor; Ligation; Neurons; Phosphoproteins; Sciatic Nerve; Signal Transduction; Snails; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

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

Platelets are cleared from circulation after a life span of 8-10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 degrees C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.

Topics: Apoptosis; Blood Platelets; Blood Preservation; Calcium; Calpain; Caspases; Culture Media, Serum-Free; Cysteine Proteinase Inhibitors; Cytochromes c; Cytoskeleton; Cytosol; Dipeptides; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gelsolin; Humans; Mitochondria; Phosphatidylserines; Platelet Aggregation; Protein-Tyrosine Kinases; src-Family Kinases; Thrombin

Human peripheral blood mononuclear cells (PBMCs) were activated using anti-CD3/CD28 (HIT3A/CD28.2) resulting in degradation of IkB alpha, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkB alpha began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkB alpha degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkB alpha levels compared to nonactivated controls. Recombinant IkB alpha was incubated with purified porcine m-calpain in the presence of 0.1% Triton X-100, and the degradation products were monitored by SDS-PAGE and sequenced. Most of the degradation products were peptides derived from calpain, but one was derived from IkB alpha cleaved between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire signal response domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. The results suggest that calpain plays an important role in IkB alpha degradation, a crucial event in T cell activation.

Topics: Amino Acid Sequence; Base Sequence; Calpain; CD28 Antigens; CD3 Complex; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Humans; I-kappa B Proteins; Kinetics; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphocytes; Molecular Sequence Data; NF-kappa B; NF-KappaB Inhibitor alpha; Oligonucleotide Probes

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimer's disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Cadaver; Calcium; Calpain; Cells, Cultured; Cytoskeleton; Dipeptides; Enzyme Activation; Enzyme Inhibitors; Humans; MAP Kinase Signaling System; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurofibrils; Neurons; Phosphorylation; Signal Transduction; Spectrin; tau Proteins

Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders.

Topics: Calpain; Cell Adhesion; Cyclic GMP; Dipeptides; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Fibronectins; Humans; Leupeptins; Mast Cells; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Protein Binding; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Tumor Cells, Cultured

Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ABCA1, the defective molecule in Tangier disease, mediates the efflux of phospholipids and cholesterol from cells to apoA-I, reversing foam cell formation. In ABCA1, we identified a sequence rich in proline, glutamic acid, serine, and threonine (PEST sequence) that enhances the degradation of ABCA1 by calpain protease and thereby controls the cell surface concentration and cholesterol efflux activity of ABCA1. In an apparent positive feedback loop, apoA-I binds ABCA1, promotes lipid efflux, inhibits calpain degradation, and leads to increased levels of ABCA1. ApoA-I infusion also increases ABCA1 in vivo. These studies reveal a novel mode of regulation of ABCA1 by PEST sequence-mediated calpain proteolysis that appears to be reversed by apolipoprotein-mediated phospholipid efflux. Inhibition of ABCA1 degradation by calpain could represent a novel therapeutic approach to increasing macrophage cholesterol efflux and decreasing atherosclerosis.

Topics: Amino Acid Sequence; Animals; Apolipoprotein A-I; Arteriosclerosis; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Biological Transport; Calpain; Cell Line; Cell Membrane; Cells, Cultured; Chickens; Cholesterol; Cross-Linking Reagents; Dipeptides; DNA; Dose-Response Relationship, Drug; Endopeptidases; Glutamic Acid; Hepatocytes; Humans; Ligands; Lipid Metabolism; Macrophages; Mice; Molecular Sequence Data; Phospholipids; Plasmids; Proline; Serine; Threonine; Time Factors; Transfection; Up-Regulation

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.

Topics: Aphidicolin; Calpain; Cell Cycle; Cell Nucleus; Chromones; Complement Activation; Complement C2; Complement C2b; Dipeptides; Enzyme Inhibitors; Flow Cytometry; HL-60 Cells; Humans; Morpholines; Nuclear Envelope; Phosphatidylinositol 3-Kinases; Phosphatidylinositols

Oxidative stress plays an important role in the development of ischemia/reperfusion (I/R)-induced apoptosis of hepatocytes. We aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1,000 micro M). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250 micro M. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked H2O2-induced procaspase-3 proteolysis and PARP cleavage without changing Bax cleavage, but partially attenuated H2O2-induced apoptosis. Calpeptin, a calpain inhibitor, did not inhibit caspase-3 activation, Bax cleavage or apoptosis. Our results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes exposed to H2O2, but not independent upon calpain. Molecular targeting of Bax cleavage may allow the development of strategies to prevent hepatic I/R injury.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Precursors; Hepatocytes; Humans; Hydrogen Peroxide; Oxidants; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Cultured mammalian PC12 or B104 cells do not instantaneously restore a plasmalemmal barrier (seal) after neurite transection, as measured using fluorescent dye probes of various sizes and saline solutions with different [Ca(2+)](o). Rather, transected cells gradually (from 15 to 60 min postseverance) exclude probes (dye molecules) of progressively smaller size. Furthermore, an inhibitor (calpeptin) of a Ca(2+)-activated cysteine protease (calpain) and antibodies or toxins to a Ca(2+)-regulated protein (synaptotagmin) and other membrane fusion proteins (syntaxin and synaptobrevin) inhibit plasmalemmal sealing. These data obtained using molecular probes on mammalian cell lines are consistent with previous data on invertebrate giant axons indicating that Ca(2+) plays many roles in the formation, accumulation, and fusion/interaction of vesicles gradually forming a seal at a site of plasmalemmal damage.

Topics: Animals; Axotomy; Calcium; Calcium-Binding Proteins; Calpain; Cell Membrane; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Extracellular Fluid; Membrane Fusion; Membrane Glycoproteins; Membrane Proteins; Nerve Regeneration; Nerve Tissue Proteins; Neurites; Neuroblastoma; PC12 Cells; Qa-SNARE Proteins; R-SNARE Proteins; Rats; Synaptotagmins; Tumor Cells, Cultured

Glutamate induces gene transcription in numerous physiological and pathological conditions. Among the glutamate-responsive transcription factors, NF-kappaB has been mainly implicated in neuronal survival and death. Recent data also suggest a role of NF-kappaB in neural development and memory formation. In non-neuronal cells, degradation of the inhibitor IkappaBalpha represents a key step in NF-kappaB activation. However, little is known of how glutamate activates NF-kappaB in neurons. To investigate the signalling cascade involved we used primary murine cerebellar granule cells. Glutamate induced a rapid reduction of IkappaBalpha levels and nuclear translocation of the NF-kappaB subunit p65. The glutamate-induced reduction of IkappaBalpha levels was blocked by the N-methyl-d-aspartate inhibitor MK801. Specific inhibitors of the proteasome, caspase 3, and the phosphoinositide 3-kinase had no effect on glutamate-induced IkappaBalpha degradation. However, inhibition of the glutamate-activated Ca2+-dependent protease calpain by calpeptin completely blocked IkappaBalpha degradation and reduced the nuclear translocation of p65. Calpeptin also partially blocked glutamate-induced cell death. Our data indicate that the Ca2+-dependent protease calpain is involved in the NF-kappaB activation in neurons in response to N-methyl-d-aspartate receptor occupancy by glutamate. NF-kappaB activation by calpain may mediate the long-term effects of glutamate on neuron survival or memory formation.

Topics: Active Transport, Cell Nucleus; Animals; Animals, Newborn; Calpain; Cell Death; Cell Differentiation; Cell Survival; Cells, Cultured; Cerebellar Cortex; Dipeptides; Down-Regulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glutamic Acid; I-kappa B Proteins; Immunohistochemistry; Memory; Mice; Nervous System; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Subunits; Receptors, N-Methyl-D-Aspartate; Signal Transduction

Our laboratory has established that local activation of calpain by a transient elevation of the free intracellular calcium concentration is crucial for the induction of growth cone (GC) formation in cultured Aplysia neurons. The mechanisms and stages in which calpain is involved in the formation of a GC are not known. We began to study these questions by determining the nature of calpain's action and the stages in which calpain activity affects the cascade of events that leads to the formation of the GC and its extension. We report that the calpain-dependent transformation of an axonal segment into a GC occurs within a narrow window of opportunity that lasts approximately 5 min. If calpain is inhibited during this window of opportunity, GC formation does not occur. Inhibition of calpain after the window of opportunity slows down the rate of lamellipodial extension but doesn't arrest it. The proteolysis of spectrin, a calpain substrate and a major component of the membrane skeleton, occurs within this window of opportunity, in agreement with the hypothesis that spectrin proteolysis is an early step in the formation of the GC. If the onset of proteolysis is deferred, spectrin remains unchanged and GC formation is compromised. We suggest that calpain participates in two different processes: it is critical for the triggering of GC formation and plays a modulatory role during the extension of the GC's lamellipodia.

Topics: Animals; Aplysia; Axotomy; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Growth Cones; Immunohistochemistry; Ionomycin; Ionophores; Nerve Regeneration; Neurons; Signal Transduction; Spectrin; Time Factors

CYP2E1-dependent mitochondrial damage, in the presence or absence of extracellular calcium, was investigated. HepG2 cells expressing CYP2E1 (E47 cells) were preloaded with arachidonic acid (AA), washed, and incubated with iron-nitrilotriacetate 1:3 complex (Fe-NTA) in minimum essential medium (MEM) (1.8mM Ca(2+)) or Ca(2+)-free MEM (SMEM). Toxicity in SMEM was CYP2E1-dependent, necrotic, and lipid peroxidation-dependent. Intracellular calcium did not significantly change during the incubation in SMEM. Mitochondrial damage preceded the loss of plasma membrane integrity and was significant at 12h of incubation, in coincidence with the toxicity. E47 cells treated with AA+Fe in MEM also showed a decline of mitochondrial membrane potential (Delta(Psi)(m)) that preceded the loss of plasma membrane integrity, but starting at earlier times, e.g., 3h than in SMEM. The decline in Delta(Psi)(m) and the toxicity in both MEM and SMEM were inhibited by alpha-tocopherol and cyclosporin A, while the calpain inhibitor calpeptin was only effective in MEM. In conclusion, oxidative damage to mitochondria and the permeability transition plays a role in the CYP2E1-dependent toxicity of Fe+AA in HepG2 cells, both in MEM and SMEM. Ca(2+) mobilization and activation of calpain contributes to the more rapid onset of mitochondrial damage in MEM, while oxidative damage and lipid peroxidation are involved in the Ca(2+)-independent later onset of mitochondrial damage.

Topics: alpha-Tocopherol; Arachidonic Acid; Calcium; Calpain; Carcinoma, Hepatocellular; Culture Media; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochrome P-450 CYP2E1; Cytosol; Dipeptides; Humans; Intracellular Membranes; Iron; Lipid Peroxidation; Liver Neoplasms; Mitochondria, Liver; Oxidative Stress; Permeability; Tumor Cells, Cultured

Retinoblastoma (Rb) is the most common intraocular malignancy of childhood. Although systemic and intrathecal chemotherapy with local and cranial radiotherapy have improved overall survival, the prognosis for patients with central nervous system involvement is still poor. We investigated the role of the transcription factor nuclear factor (NF)-kappaB, which promotes cell survival in several other models, in the pathophysiology of Rb. The human Rb cell lines Y79 and WERI-Rb1 were treated with the cell permeable peptide SN50, that specifically inhibits the transcriptional activity of NF-kappaB by blocking its translocation into the nucleus. We found that NF-kappaB inhibition up-regulated Bax; down-regulated the anti-apoptotic proteins Bcl-2, A1, and cIAP-2; and induced loss of the mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis in Rb cells. Inhibition of the p38 kinase sensitized cells to SN50-induced cell death, whereas insulin-like growth factor-1 activated NF-kappaB and attenuated the proapoptotic effect of SN50. Finally, NF-kappaB inhibition sensitized Rb cells to doxorubicin. In conclusion, inhibition of NF-kappaB activity in Rb cells leads to loss of mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis. Therapeutic strategies targeting NF-kappaB could be beneficial in the clinical management of Rb, either alone or in combination with conventional chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Calpain; Caspase 3; Caspases; Cell Survival; Dipeptides; DNA; Doxorubicin; Enzyme Inhibitors; Humans; Imidazoles; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Oligonucleotides, Antisense; p38 Mitogen-Activated Protein Kinases; Peptides; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma; Tumor Cells, Cultured

We have investigated a novel function of calpeptin, a commonly used inhibitor of calpain, in the production of intracellular reactive oxygen species (ROS) in Swiss 3T3 fibroblasts. Calpeptin induced a rapid increase of intracellular ROS by a dose-dependent manner, with a maximal increase at 10 min, which was inhibited by ROS scavengers, catalase and 2-MPG. However, other calpain inhibitors, E64d and N-acetyl-Leu-Leu-Nle-CHO (ALLN), had no effect on the level of intracellular ROS, indicating that calpain was not involved in the ROS production by calpeptin. The role of Rho in the ROS production by calpain was studied by scrape-loading of C3 transferase. C3 transferase, which inhibited stress fiber formation by calpeptin, had no effect on the ROS production in response to calpeptin, suggesting that Rho was not involved in the ROS production by calpeptin. But the elevation of intracellular ROS was inhibited by mepacrine, a phospholipase A2 inhibitor. In addition, scavenging intracellular ROS by the incubation with catalase and 2-MPG had no effect on the stress fiber formation by calpeptin. These results suggested that calpeptin stimulated the production of intracellular ROS and stress fiber formation by independent mechanisms.

Topics: 3T3 Cells; Animals; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Intracellular Fluid; Mice; Phospholipases A; Phospholipases A2; Reactive Oxygen Species; rho GTP-Binding Proteins

The cytokine tumor necrosis factor (TNF)alpha induces caspase-dependent cell death in a subset of tumor cells. In this report, we show a novel suppressive effect of calpeptin, a calpain inhibitor, on TNFalpha-induced cell death and accumulation of p53 in L929 mouse fibrosarcoma. Exposure to 10 ng/ml TNFalpha induced cell death in >50% of L929 cells within 12 h and stimulated accumulation of p53 (8-fold). Preincubation of cells with calpeptin blocked both TNFalpha-induced cell death and accumulation of p53 as examined with Western blot. TNFalpha-induced accumulation of p53 was in part contributed by increase of p53 mRNA level (2.2-fold) in a calpeptin-insensitive manner. Interestingly, other calpain inhibitors tested did not show these effects like calpeptin and TNFalpha treatment did not increase apparent calpain activity in L929 cells, suggesting that calpeptin may have another function besides targeting calpain. Expression of dominant negative mutant p53Val(135) reduced the incidence of TNFalpha-mediated cell death. Taken together, our findings suggest that TNFalpha induces calpeptin-dependent, but calpain-independent accumulation of p53 protein as a necessary step leading to death in L929 cells.

Topics: Animals; bcl-2-Associated X Protein; Blotting, Northern; Blotting, Western; Calpain; Cell Death; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Dipeptides; Genes, Dominant; Luciferases; Mice; Mutation; Nuclear Proteins; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Time Factors; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins were phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was estimated at 6 microM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.

Topics: Adult; Blood Platelets; Calmodulin-Binding Proteins; Calpain; Cytoskeletal Proteins; Dipeptides; Humans; In Vitro Techniques; Kinetics; Phosphorylation; Platelet Activation; Platelet Aggregation; Protein Kinase C; Recombinant Proteins; Regression Analysis; Staurosporine; Thrombin

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.

Topics: Amino Acid Sequence; Apoptosis; Arginine; bcl-2 Homologous Antagonist-Killer Protein; BH3 Interacting Domain Death Agonist Protein; Calcium; Calpain; Carrier Proteins; Cathepsin L; Cathepsins; Cisplatin; Cysteine Endopeptidases; Cytochrome c Group; Dipeptides; Enzyme Activation; Glycine; Humans; Intracellular Membranes; Membrane Potentials; Membrane Proteins; Mitochondria; Molecular Sequence Data; Protein Processing, Post-Translational; Signal Transduction; Time Factors; Tumor Cells, Cultured

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Line; Cell Transformation, Neoplastic; Chelating Agents; Cysteine Endopeptidases; Dipeptides; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionophores; Lymphoma; Mammary Neoplasms, Experimental; Mice; Multienzyme Complexes; Mutation; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Rats

In murine L cells, treatment with calpeptin or calpain inhibitor III increased Abeta42, but not Abeta40, secretion in a dose-dependent fashion. This correlated with an increase in the levels of amyloid precursor protein (APP) carboxyl-terminal fragments (CTFs). Immunoprecipitation with novel mAbs directed against the carboxyl-terminus of APP or specific for the beta-cleaved CTF showed that generation of both alpha- and beta-cleaved CTFs increase proportionately following inhibition of calpains. Pulse-chase metabolic labeling confirmed that inhibiting calpains increases the production of alpha- and beta-cleaved APP metabolites. Immunolabeling showed greater betaCTF signal in calpeptin-treated cells, primarily in small vesicular compartments that were shown to be predominantly endosomal by colocalization with early endosomal antigen 1. A second mAb, which recognizes an extracellular/luminal epitope found on both APP and betaCTFs, gave more cell surface labeling of calpeptin-treated cells than control cells. Quantitative binding of this antibody confirmed that inhibiting calpains caused a partial redistribution of APP to the cell surface. These results demonstrate that 1) calpain inhibition results in a partial redistribution of APP to the cell surface, 2) this redistribution leads to an increase in both alpha- and beta-cleavage without changing the ratio of alphaCTFs/betaCTFs, and 3) the bulk of the betaCTFs in the cell are within early endosomes, confirming the importance of this compartment in APP processing.

Topics: Amyloid beta-Protein Precursor; Animals; Antibodies, Monoclonal; Blotting, Western; Calpain; Cell Line; Cell Membrane; Dipeptides; Dose-Response Relationship, Drug; Endosomes; Enzyme-Linked Immunosorbent Assay; Humans; Hydrogen-Ion Concentration; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Models, Genetic; Mutation; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Time Factors; Tumor Cells, Cultured

Although global ischemia induces troponin I (TnI) degradation, regional ischemia does not. We hypothesized that this disparity is related to preload-induced proteolysis, which varies as a function of the amount of myocardium at risk of ischemia.. Isolated rat hearts were buffer-perfused at controlled levels of preload. Increasing preload to 25 mm Hg in the absence of ischemia produced pronounced TnI degradation (27 kDa versus 31 kDa bands: 16.4 +/- 3.6% versus 4.7 +/- 1.9% in immediately excised controls, P<0.05). TnI degradation could be blocked by preventing the activation of endogenous calpains with 25 micromol/L calpeptin (4.3 +/- 0.6%). This improved function, with left ventricular systolic pressure increasing from 103 +/- 4 mm Hg to 137 +/- 7 mm Hg (P<0.05). Eliminating elevations in preload after global ischemia-induced stunning also prevented TnI degradation.. Calpain-mediated TnI proteolysis can be dissociated from stunning and arises from elevations in preload rather than ischemia. This raises the possibility that ongoing preload-induced TnI degradation could impair myocardial function long-term.

Topics: Animals; Blood Pressure; Calpain; Cardiac Pacing, Artificial; Cysteine Proteinase Inhibitors; Dipeptides; Heart; Hemodynamics; In Vitro Techniques; Myocardial Ischemia; Myocardium; Rats; Rats, Sprague-Dawley; Troponin I; Ventricular Function, Left

Apoptosis participates in the development of the nervous system and in neurodegeneration. The aim of this study was to investigate the mechanisms of detachment of neuronal cells from the extracellular matrix (ECM) during apoptosis. Detachment of Ntera2 neuronal cells was accompanied by decreased surface expression of the beta1 integrin and redistribution of proteins from focal adhesions (FA). FA proteins were cleaved in a discrete sequence: p130cas, then paxillin, then talin. Caspase inhibition prevented detachment and cleavage of paxillin and p130cas, whilst calpain inhibition blocked talin cleavage. Neuronal cells therefore detach as a result of redistribution and caspase-dependent cleavage of focal adhesion proteins. Cleavage occurs sequentially such that critical ECM-integrin survival signalling cascades are severed before disruption of focal adhesion-cytoskeletal links.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase Inhibitors; Caspases; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Extracellular Matrix; Focal Adhesions; Neurons; Paxillin; Phosphoproteins; Proteins; Retinoblastoma-Like Protein p130; Talin

T cells treated with the drug etoposide undergo apoptotic death characterized by early evidence of nuclear damage followed by loss of mitochondrial integrity and cell lysis. Calpains and caspases are cytoplasmic proteases and there is increasing evidence of cross-talk between these proteases in death pathways. In this study we have investigated the role of calpain, in etoposide-triggered apoptosis in the 2B4 murine T cell hybridoma. Cell permeable inhibitors of calpain, ALLnM, E64 and calpeptin that block Fas ligand-Fas-mediated death in T cells, blocked etoposide-induced nuclear damage, loss of mitochondrial integrity and cell lysis. A broad spectrum peptide inhibitor of caspases, ZVAD-fmk, partially blocked nuclear damage but poorly inhibited mitochondrial damage or cell lysis triggered by etoposide. Etoposide-induced expression of the cleaved, proteolytically active form of caspase 3, and DEVD-ase activity, detected prior to nuclear damage, were blocked in the presence of calpain inhibitors. Collectively, these data describe a role for calpain in regulating etoposide-induced apoptosis via a caspase-dependent pathway in T cells.

Topics: Animals; Apoptosis; Calpain; Caspases; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Etoposide; Fas Ligand Protein; Hybridomas; Leucine; Membrane Glycoproteins; Mice; T-Lymphocytes

Doublecortin-like kinase (DCLK) is widely expressed in postmitotic neurons throughout the embryonic nervous system. DCLK consists of an N-terminal doublecortin domain, responsible for its localization to microtubules, and a C-terminal serine-threonine kinase domain. Here we report that DCLK is a physiological substrate for the cysteine protease calpain. Cleavage of DCLK by calpain severs the kinase domain from its microtubule anchorage domain and releases it into the cytoplasm. The isolated kinase domain retains catalytic activity and is structurally similar to CPG16, a second product of the DCLK gene expressed in the adult brain that lacks the doublecortin domain. We propose that in neurons cleavage of DCLK by calpain represents a calcium responsive mechanism to regulate localization of the DCLK kinase domain.

Topics: Animals; Blotting, Western; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Cell Line; Cells, Cultured; Cytoplasm; Cytosol; Dipeptides; Dose-Response Relationship, Drug; Doublecortin Domain Proteins; Doublecortin-Like Kinases; Glutathione Transferase; Humans; Intracellular Signaling Peptides and Proteins; Mice; Microtubule-Associated Proteins; Microtubules; Models, Genetic; Nerve Tissue Proteins; Neurons; Neuropeptides; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Recombinant Fusion Proteins; Recombinant Proteins; Subcellular Fractions; Time Factors; Transfection

Ca2+ influx is one of the main causative events in hypoxic PC12 cell death, because an extracellular Ca2+ chelator, ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited and Ca2+ ionophore A23187 mimicked the hypoxic cell death. The hypoxic cell death was markedly prevented by a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) as well as a calpain inhibitor, calpeptin, as assessed by nuclear staining with Hoechst 33258 and lactate dehydrogenase release. The processing of procaspase-3 was inhibited by z-VAD-FMK, but not by calpeptin. In contrast, z-VAD-FMK failed to block the proteolytic cleavage of fodrin-alpha, a preferential substrate for calpain. On the other hand, degradation of actin and fodrin-alpha was prevented by calpeptin but not by z-VAD-FMK. In addition, not only protein kinase C (PKC)-alpha but also PKC-delta were cleaved to generate approximately 46 kDa fragments. The PKC fragmentation was inhibited by calpeptin but not by z-VAD-FMK. These findings suggest that the extracellular Ca2+ influx induced by hypoxic stress activates calpain, resulting in the degradation of cytoskeletal proteins and generation of PKC fragments almost independently of caspase activation. Therefore, calpain may play an important role in hypoxic PC12 cell death.

Topics: Animals; Calcimycin; Calcium; Calpain; Caspases; Cell Death; Chelating Agents; Cysteine Proteinase Inhibitors; Cytoskeleton; Dipeptides; Egtazic Acid; Hypoxia-Ischemia, Brain; Ionophores; Isoenzymes; Nerve Degeneration; PC12 Cells; Peptide Hydrolases; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Rats

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.

Topics: Amino Acid Sequence; Animals; Autoantigens; Calpain; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Dystrophin-Associated Proteins; Enzyme Activation; Enzyme Inhibitors; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Membrane Proteins; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Protein Binding; Protein Precursors; Protein Tyrosine Phosphatases; Rats; Receptor-Like Protein Tyrosine Phosphatases, Class 2; Receptor-Like Protein Tyrosine Phosphatases, Class 8; Secretory Vesicles; Tumor Cells, Cultured; Utrophin

Platelet-associated Bruton's tyrosine kinase (Btk) was completely cleaved if treated with calcium ionophore A23187 with appearance of a proteolytic product of 27 kDa size. Aggregation with thrombin also induced about 10% degradation of Btk after 30 min. Calpain inhibitors prevented Btk degradation in both. The proteolytic products of the Wiskott-Aldrich syndrome protein (WASP), a calpain and Btk substrate, and the 27 kDa degradation product of Btk did not redistribute to the Triton-insoluble cytoskeleton in thrombin-aggregated platelets, in contrast to the uncleaved proteins. The degradation of Btk and WASP was independent of their tyrosine phosphorylation status. These results indicate that Btk is an endogenous substrate for calpain, the cleavage of which may have functional consequences in long-term post-aggregation events in platelets.

Topics: Agammaglobulinaemia Tyrosine Kinase; Blood Platelets; Calcimycin; Calcium; Calpain; Cells, Cultured; Dipeptides; Humans; Ionophores; Octoxynol; Protein-Tyrosine Kinases; Proteins; Thrombin; Wiskott-Aldrich Syndrome Protein

The neurotoxic phospholipase A(2), beta-bungarotoxin (beta-BuTx), is a component of the snake venom from the Taiwanese banded krait Bungarus multicinctus. beta-BuTx affects presynaptic nerve terminal function of the neuromuscular junction and induces widespread neuronal cell death throughout the mammalian and avian CNS. To analyse the initial events of beta-BuTx-mediated cell death, the toxin was applied to cultured rat hippocampal neurons where it induced neuronal cell death in a concentration-dependent manner (EC(50) approximately equal to 5 x 10(-13) M) within 24 h. Fluorescence labelled beta-BuTx was completely incorporated by neurons within < 10 min. Binding and uptake of beta-BuTx, as well as induction of cell death, were efficiently antagonized by preincubation with dendrotoxin I, a blocker of voltage-gated potassium channels devoid of phospholipase activity. Binding of beta-BuTx was selective for neurofilament-positive cells. As evident from intense annexin-V and TUNEL stainings, application of beta-BuTx induced apoptotic cell death exclusively in neurons, leaving astrocytes unaffected. No evidence was obtained for any contribution of either caspases or calpains to beta-BuTx-induced apoptosis, consistent with the inability of the inhibitors Z-Asp-DCB and calpeptin, respectively, to protect neurons from beta-BuTx-induced cell death. These observations indicate that induction of cell death by beta-BuTx comprises several successive phases: (i) binding to neuronal potassium channels is the initial event, followed by (ii) internalization and (iii) induction of apoptotic cell death via a caspase-independent pathway.

Topics: Animals; Annexin A5; Apoptosis; Bungarotoxins; Calpain; Caspase Inhibitors; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Elapid Venoms; Female; Hippocampus; In Situ Nick-End Labeling; Neurons; Neurotoxins; Phospholipases A; Pregnancy; Rats; Rats, Wistar; Receptors, Cell Surface; Signal Transduction

Moloney murine leukemia virus (MoMuLV)-ts1-mediated neuronal degeneration in mice is likely due to loss of glial support and release of inflammatory cytokines and neurotoxins from surrounding ts1-infected glial cells including astrocytes. NF-kappaB is a transcription factor that participates in the transcriptional activation of a variety of immune and inflammatory genes. We investigated whether ts1 activates NF-kappaB in astrocytes and examined the mechanism(s) responsible for the activation of NF-kappaB by ts1 infection in vitro. Here we present evidence that ts1 infection of astrocytes in vitro activates NF-kappaB by enhanced proteolysis of the NF-kappaB inhibitors, IkappaBalpha and IkappaBbeta. In in vitro studies using protease inhibitors, IkappaBalpha proteolysis in ts1-infected astrocytes was significantly blocked by a specific calpain inhibitor calpeptin but not by MG-132, a specific proteasome inhibitor, whereas rapid IkappaBbeta proteolysis was blocked by MG-132. Furthermore, treatment with MG-132 increased levels of multiubiquitinated IkappaBbeta protein in ts1-infected astrocytes. These results indicate that the calpain proteolysis is a major mechanism of IkappaBalpha proteolysis in ts1-infected astrocytes. Additionally, ts1 infection of astrocytes in vitro increased expression of inducible nitric oxide synthase (iNOS), a NF-kappaB-dependent gene product. Our results suggest that NF-kappaB activation in ts1-infected astrocytes is mediated by enhanced proteolysis of IkappaBalpha and IkappaBbeta through two different proteolytic pathways, the calpain and ubiquitin-proteasome pathways, resulting in increased expression of iNOS, a NF-kappaB-dependent gene.

Topics: Active Transport, Cell Nucleus; Animals; Astrocytes; Calpain; Cells, Cultured; Cysteine Endopeptidases; Dipeptides; DNA-Binding Proteins; Enzyme Induction; Gene Expression Regulation, Viral; I-kappa B Proteins; Leupeptins; Ligases; Mice; Moloney murine leukemia virus; Multienzyme Complexes; Nerve Tissue Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protease Inhibitors; Proteasome Endopeptidase Complex; Ubiquitin

Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.

Topics: Actins; Androstadienes; Animals; Calpain; Cell Adhesion; Cell Size; Chromones; Dipeptides; Enzyme Activation; Estrenes; Extracellular Matrix; Fibrinogen; Isoenzymes; Morpholines; Muscle, Smooth, Vascular; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Phosphoinositide-3 Kinase Inhibitors; Protein Binding; Protein Subunits; Pyrrolidinones; Swine; Type C Phospholipases; Wortmannin

Mutations of Wiskott-Aldrich syndrome protein (WASP) underlie the severe thrombocytopenia and immunodeficiency of the Wiskott-Aldrich syndrome. WASP, a specific blood cell protein, and its close homologue, the broadly distributed N-WASP, function in dynamic actin polymerization processes. Here it is demonstrated that N-WASP is expressed along with WASP, albeit at low levels, in human blood cells. The presence of approximately 160 nmol/L rapidly acting N-WASP molecules may explain the normal capacity of WASP-negative patient platelets for early agonist-induced aggregation and filopodia formation. Ex vivo experiments revealed a significant difference between WASP and N-WASP in sensitivity to calpain, the Ca++-dependent protease activated in agonist-stimulated platelets. Through the use of a series of calpain-containing broken cell systems, it is shown that WASP is cleaved in a Ca++-dependent reaction inhibitable by calpeptin and E64d and that N-WASP is not cleaved, suggesting that the cleavage of WASP by calpain functions in normal platelets as part of a Ca++-dependent switch mechanism that terminates the surface projection phase of blood cell activation processes.

Topics: Blood Platelets; Calcium; Calpain; Cell-Free System; Dipeptides; Female; HeLa Cells; Humans; Male; Nerve Tissue Proteins; Platelet Activation; Proteins; Pseudopodia; Sensitivity and Specificity; Substrate Specificity; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein; Wiskott-Aldrich Syndrome Protein, Neuronal

Prolonged treatment of rat adipocytes with TNF alpha increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (G(i)). Separately, down-regulation of G(i) by prolonged treatment with an A(1)-adenosine receptor agonist, N(6)-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNF alpha and PIA-mediated G(i) down-regulation and stimulation of lipolysis, we used the protease inhibitors lactacystin (proteasome inhibitor) and calpeptin (calpain inhibitor). Rat adipocytes were preincubated for 1 h with lactacystin (10 microM) or calpeptin (50 microM), before 30-h treatment with either TNF alpha (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of alpha-subunits of G(i)1 and G(i)2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF alpha and PIA stimulated lipolysis approximately 2-fold and caused G(i) down-regulation. Although neither lactacystin nor calpeptin affected basal lipolysis, lactacystin completely inhibited both TNF alpha and PIA-stimulated lipolysis (the 50% inhibitory concentration was approximately 2 microM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNF alpha-induced G(i) down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF alpha and PIA is secondary to G(i) down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.

Topics: Acetylcysteine; Adipocytes; Animals; Calpain; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Down-Regulation; GTP-Binding Protein alpha Subunits, Gi-Go; Lipolysis; Male; Multienzyme Complexes; Phenylisopropyladenosine; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

The effects of calpain inhibitors on the total number of acetylcholine receptors (AChRs) on cultured rat myotubes and on the stability of AChR clusters in these myotubes were investigated. The degradation rate of total AChRs labeled with (125)I-alpha-bungarotoxin was assessed from radioactivity remaining in the myotubes as a function of time. Treatment with calpain inhibitors resulted in a two- to three-fold increase in the half-life of total AChRs. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation. The rate loss of AChRs from the clusters was assessed by measuring the loss of fluorescence intensity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatment with calpain inhibitors resulted in twofold increases in cluster half-life. Thus, there was generally no change in total surface receptors with the calpain inhibitors, whereas cluster half-life was substantially increased. Furthermore, with a low dose of calpeptin there was no change in turnover of total cellular AChRs, whereas cluster half-life was doubled. Taken together, these results suggest that the increased half-life of clusters produced by the calpain inhibitors may be due to retardation of the lateral movement from AChRs in the clusters.

Topics: Animals; Animals, Newborn; Bungarotoxins; Calpain; Cells, Cultured; Dipeptides; Myofibrils; Rats; Receptors, Cholinergic

We have followed the in vitro degradation of rat histidine decarboxylase in a reconstituted system, containing only rat histidine decarboxylase (obtained by in vitro transcription and translation), calcium ions in the millimolar range of concentrations, and m-calpain. Under the experimental conditions used, m-calpain quickly and efficiently degraded rat histidine decarboxylase, giving rise to a major proteolytic band of 29 kDa. In a conventional in vitro degradation system containing rabbit reticulocytes supplemented with calcium ions, there was also an intense proteolysis of rat histidine decarboxylase, strongly inhibited in the presence of calpeptin, a highly specific calpain inhibitor.

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Histidine Decarboxylase; Kinetics; Peptide Fragments; Rats; Recombinant Proteins; Reticulocytes; Substrate Specificity

Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O(2)-95% N(2)-5% CO(2)) in the presence and absence of calpain inhibitor I or calpeptin, after which endothelial nitric oxide synthase (eNOS) activity and protein content were assayed. Exposure to hypoxia decreased eNOS activity but not eNOS protein content. Both calpain inhibitor I and calpeptin prevented the hypoxic decrease of eNOS activity. Incubation of calpain with total membrane preparations of PAEC caused dose-dependent decreases in eNOS activity independent of changes in eNOS protein content. Exposure of PAEC to hypoxia also caused time-dependent decreases of heat shock protein 90 (HSP90) that were prevented by calpain inhibitor I and calpeptin. Moreover, the HSP90 content in anti-eNOS antibody-induced immunoprecipitates from hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the decrease of eNOS activity in these immunoprecipitates. Incubation of PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the hypoxic decrease of eNOS activity. These results indicate that the hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease in HSP90 caused by calpain activation.

Topics: Animals; Calpain; Caveolin 1; Caveolins; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Endothelium, Vascular; HSP90 Heat-Shock Proteins; Hypoxia; Membrane Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Phosphorylation; Pulmonary Artery; Serine; Swine; Tyrosine

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.

Topics: Apoptosis; Base Sequence; bcl-2-Associated X Protein; Calpain; Cytochrome c Group; Dipeptides; DNA Primers; Enzyme Activation; Etoposide; Humans; Hydrolysis; Jurkat Cells; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.

Topics: 5'-Nucleotidase; Antibodies, Monoclonal; Calpain; Cell Adhesion; Cell Communication; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Endothelium, Vascular; Humans; Lymphocyte Function-Associated Antigen-1; Lymphocytes; Receptor Aggregation; Umbilical Veins; Up-Regulation

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.

Topics: Adult; Antibodies, Monoclonal; Calpain; CD3 Complex; Cell Adhesion; Cell Movement; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Fibronectins; Humans; Integrin beta1; Lymphocyte Activation; T-Lymphocytes; Tetradecanoylphorbol Acetate

Many studies have demonstrated that m-calpain was implicated in cell membrane reorganization-related phenomena during fusion via a regulation by calpastatin, the specific Ca2+-dependent proteolytic inhibitor. However, the real biological role of this protease is unclear because many targeted proteins are still unknown. Using different digestion experiments we have demonstrated that desmin, vimentin, talin, and fibronectin represent very good substrates for this proteinase capable of cleaving them in fragments which are immediately degraded by other enzymatic systems. Concerning intermediate filaments, we showed that during the phenomenon of fusion, the amount of desmin was significantly reduced while the concentration of vimentin presented a steady level. On the other hand, we have conducted biological assays on cultured myoblasts supplemented by exogenous factors such as calpain inhibitors or antisense oligonucleotides capable of stimulating or inhibiting m-calpain activity. The effect of such factors on fusion and concomitantly on the targeted substrates was analyzed and quantified. When m-calpain activity and myoblast fusion were prevented by addition of calpain inhibitors entering the cells, the amounts of desmin, talin, and fibronectin were increased, whereas the amount of vimentin was unchanged. Using antisense strategy, similar results were obtained. In addition, when the phenomenon of fusion was enhanced by preventing calpastatin synthesis, the amounts of desmin, talin, and fibronectin were significantly reduced. Taken together, these results support the hypothesis that m-calpain is involved in myoblast fusion by cleaving certain proteins identified here. This cleavage could modify membrane and cytoskeleton organization for the myoblasts to fuse.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Extracts; Cell Fusion; Cells, Cultured; Cysteine Proteinase Inhibitors; Desmin; Dipeptides; Octoxynol; Oligonucleotides, Antisense; Rats; Rats, Wistar; Substrate Specificity; Vimentin

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase 3; Caspases; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; HL-60 Cells; Humans; Oligopeptides

Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of protein-tyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.

Topics: 3T3 Cells; Animals; Binding Sites; Calpain; Cell Membrane; Cross Reactions; Cysteine; Cytoskeletal Proteins; Dipeptides; Fibroblasts; GTP Phosphohydrolases; GTP-Binding Proteins; Isoenzymes; Mice; Microscopy, Fluorescence; Paxillin; Phosphoproteins; Protein Tyrosine Phosphatases; rho GTP-Binding Proteins; Structure-Activity Relationship

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calcimycin; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioma; Hydroxyl Radical; Interferon-gamma; Ionophores; Rats; Tumor Cells, Cultured

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.

Topics: Blood Proteins; Calcium; Calpain; Dipeptides; Enzyme Activation; Humans; Ionophores; Phosphorylation; Platelet Activation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases

Striatal enriched phosphatase (STEP) is a family of protein tyrosine phosphatases enriched within the CNS. A member of this family, STEP61, is a membrane-associated protein located in postsynaptic densities of striatal neurons. In this study, we demonstrate that STEP61, is cleaved into smaller isoforms. To clarify the mechanism of cleavage, STEP61 was transiently expressed in NT2/D1 neuronal precursor cells. Exposure of transfected cells to the calcium ionophore, A23187, or to thapsigargin resulted in the rapid cleavage of STEP61. Pretreatment with the calpain inhibitor, calpeptin, or EGTA prevented proteolysis. One of the cleavage products has a relative molecular mass of 33 kDa (STEP33). A protein with the identical mobility is detected following calpain treatment of STEP61 fusion protein or postsynaptic densities purified from rat striatum. Exposure of primary neuronal cultures to glutamate also led to a significant increase in the concentration of a low molecular weight form of STEP. Taken together, these results suggest that in response to a rapid influx of calcium, STEP61, is proteolytically cleaved by calpain, leading to the release of a smaller isoform. This model may explain the rapid appearance of STEP33 in response to transient hypoxia-ischemia in the brain as cells attempt to counter the increase in tyrosine phosphorylation levels following neuronal insults.

Topics: Animals; Calcimycin; Calcium; Calpain; Dipeptides; Egtazic Acid; Female; Glutamic Acid; Ionophores; Isoenzymes; Kinetics; Molecular Weight; Protein Tyrosine Phosphatases; Protein Tyrosine Phosphatases, Non-Receptor; Rats; Rats, Long-Evans; Thapsigargin; Transfection

Acute renal failure was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function parameters such as blood urea nitrogen, plasma creatinine, creatinine clearance, urine flow and urinary osmolality were measured to test the effectiveness of drugs. Renal function in untreated acute renal failure rats markedly decreased at 24 h after reperfusion. The administration of PSI, N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinal, a proteasome inhibitor, at a dose of 1 mg/kg before the occlusion abolished the decreases in the renal function of acute renal failure rats. Calpeptin (1 mg/kg), a calpain inhibitor, attenuated the deterioration of renal function to the same extent as 0.1 mg/kg PSI, but no significant difference was observed between the untreated and calpeptin-treated acute renal failure groups. Histopathological examination of the kidney of untreated acute renal failure rats revealed severe lesions, such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion, all of which were significantly suppressed by PSI (1 mg/kg) treatment. In contrast, calpeptin, at the same dose, was ineffective against the development of renal lesions. These results suggest that proteasome participates in the pathogenesis of ischemic acute renal failure. Thus, proteasome may be a potential target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent on ischemia/reperfusion.

Topics: Acute Kidney Injury; Animals; Calpain; Cysteine Endopeptidases; Dipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ischemia; Kidney; Male; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley

Intracellular free Ca2+ and free radicals are increased following spinal cord injury (SCI). These can activate calpain to degrade cytoskeletal proteins leading to apoptotic and necrotic cell death. Primary injury triggers a cascade of secondary injury, which spreads to rostral and caudal areas. We tested calpain involvement in apoptosis in five 1-cm segments of rat spinal cord with injury (40 g-cm) induced at T12 by weight-drop. Animals were immediately treated with calpeptin (250 micrograms/kg) and methylprednisolone (165 mg/kg) and sacrificed at 48 hr. Untreated SCI rats manifested 68-kD neurofilament protein (NFP) degradation (indicating calpain activity), and internucleosomal DNA fragmentation (indicating apoptosis). Both calpain activity and apoptosis were highest in the lesion, and decreased with increasing distance from the lesion. Treatment decreased 68-kD NFP degradation with reduction in apoptosis in all five areas. Thus, calpeptin and methylprednisolone are found to be neuroprotective in SCI.

Topics: Animals; Apoptosis; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Methylprednisolone; Neurofilament Proteins; Neuroprotective Agents; Rats; Spinal Cord Injuries; Thoracic Vertebrae

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.

Topics: Calcium-Binding Proteins; Calpain; Cell Adhesion Molecules; Cell Division; Dipeptides; DNA; Flow Cytometry; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Isomerism; Muscle, Smooth, Vascular; Oligonucleotides, Antisense; Protease Inhibitors; Protein-Tyrosine Kinases

Calpains have been previously shown to regulate AMPA receptor properties by producing partial truncation of the C-terminal domains of several receptor subunits. We now report that NMDA receptor subunits, in particular NR2 subunits, are also subjected to calpain-mediated truncation. Treatment of synaptic membranes with calpain I resulted in truncation of both NR1 and NR2 subunits, with the appearance of NR2 species with lower mol.wt. than native subunits, but still recognized by antibodies directed at the C-terminal domain. This treatment did not modify the binding of several ligands of the NMDA receptors, such as glutamate, glycine or TCP. Incubation of thin frozen-thawed brain sections with calcium resulted in calpain-mediated selective degradation of NR2 subunits, as truncation into smaller fragments was totally blocked by calpain inhibitors. Under the same conditions, TCP binding to sections was decreased by about 50%, an effect also blocked by calpain inhibitors. Treatment of hippocampal slices in culture with the excitotoxin, kainic acid, also produced calpain-mediated truncation of the C-terminal domain of NR2 but not NR1 subunits of the NMDA receptors. The results indicate that calpain activation produces several modifications of NMDA receptors, including the truncation of the C-terminal domain of NR2 subunits, and changes in channel binding properties. They suggest that calpain-mediated regulation of NMDA receptors might represent a feed-back regulation of the receptors which could be used to limit receptor activation.

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Excitatory Amino Acid Agonists; Freezing; Glutamic Acid; Hippocampus; Ion Channel Gating; Kainic Acid; Neuronal Plasticity; Organ Culture Techniques; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes

The emergence of a neuronal growth cone from a transected axon is a necessary step in the sequence of events that leads to successful regeneration. Yet, the molecular mechanisms underlying its formation after axotomy are unknown. In this study, we show by real time imaging of the free intracellular Ca2+ concentration, of proteolytic activity, and of growth cone formation that the activation of localized and transient Ca2+-dependent proteolysis is a necessary step in the cascade of events that leads to growth cone formation. Inhibition of this proteolytic activity by calpeptin, a calpain inhibitor, abolishes growth cone formation. We suggest that calpain plays a central role in the reorganization of the axon's cytoskeleton during its transition from a stable differentiated structure into a dynamically extending growth cone.

Topics: Animals; Aplysia; Axotomy; Calcium; Calpain; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Membrane Proteins; Microscopy, Video; Nerve Regeneration; Neurites; Neurons; Spectrin; Time Factors

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.

Topics: Alanine; Amino Acid Sequence; Apoptosis; Aspartic Acid; bcl-2-Associated X Protein; Binding Sites; Calpain; Camptothecin; Cell Death; Cell Extracts; Cell Membrane; Cysteine Proteinase Inhibitors; Dipeptides; HL-60 Cells; Humans; Hydrolysis; Leucine; Molecular Sequence Data; Mutation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Substrate Specificity

Programmed cell death invariably requires the activation of proteolytic cascades that are not yet well defined but are initiated after apical caspase activation. We provide evidence that calpains and the proteasome function synergistically downstream of caspases to assist the constitutive apoptotic program of aging neutrophils, which plays an important role in resolution of inflammatory responses. Inhibitor studies indicated that "tethering" of preapoptotic senescent neutrophils to human macrophages required caspase activity. However, the development of morphological features characteristic of apoptosis, including nuclear morphology, PS exposure, surface protein shedding, and the capacity to be ingested by macrophages, required the downstream action of either calpains or the proteasome. Calpain activities were constitutively active in freshly isolated neutrophils and responsible for rearrangements in the protein composition and structure of the plasmalemmal cytoskeleton as they aged in culture and underwent apoptosis. This included a dissociation of protein(s) from F-actin, a candidate mechanism for increased susceptibility to cleavage, and a loss in immunodetectable alpha-actinin and ezrin, two actin-binding, membrane-anchoring proteins. These results clarify roles for different classes of proteases in a physiologically important form of constitutive apoptosis.

Topics: Actinin; Actins; Apoptosis; Calpain; Caspases; Cell Communication; Cells, Cultured; Cellular Senescence; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Drug Synergism; Humans; Leupeptins; Multienzyme Complexes; Neutrophils; Phosphoproteins; Proteasome Endopeptidase Complex

In thrombin-stimulated platelets alpha IIb beta 3 integrin engagement triggers both phosphatidylinositol 3',4'-bisphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dose-dependent inhibition of phosphatidylinositol 3,4-bisphosphate synthesis by calpeptin from 50 micrograms/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated alpha IIb beta 3 integrin, did not induce additive effects on phosphatidylinositol 3,4-bisphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptin-treated platelets. These data indicate that calpains are involved in the regulation of alpha IIb beta 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.

Topics: Biological Transport; Blood Platelets; Calpain; Cytoskeleton; Dipeptides; Humans; Integrins; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Thrombin

Inositol polyphosphate 4-phosphatase (4-phosphatase), an enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, was shown to be a substrate for the calcium-dependent protease calpain in vitro and in stimulated human platelets. Stimulation of platelets with the calcium ionophore, A23187, resulted in complete proteolysis of 4-phosphatase and a 75% reduction in enzyme activity. Thrombin stimulation of platelets resulted in partial proteolysis of 4-phosphatase and a 41% reduction in enzyme activity (n = 8, range of 36-51%). In addition, preincubation with the calpain inhibitor, calpeptin, suppressed the accumulation of phosphatidylinositol 3, 4-bisphosphate in thrombin-stimulated platelets by 36% (n = 2, range = 35-37%). These data suggest that the calpain-mediated inhibition of 4-phosphatase is involved in the phosphatidylinositol 3, 4-bisphosphate accumulation in thrombin-stimulated platelets.

Topics: Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Humans; Phosphatidylinositol Phosphates; Phosphoric Monoester Hydrolases; Platelet Activation; Substrate Specificity; Thrombin

Intracellular calcium overload of guinea pig cardiomyocytes is accompanied by troponin T cross-linking, which is revealed by changes in immunoreactivity of anti-troponin T antibodies. We presently investigated whether the same process is detectable in the human heart. Immunohistochemistry shows myofibrillar staining with BN-59 anti-troponin T antibody with rare cardiomyocytes in samples obtained at surgery, whereas approximately 50% of myocytes are labeled in heart samples taken at autopsy within 3 hours of death, and every cardiomyocyte is stained after exposure of biopsy sections to 10 mmol/L calcium. Western blot analysis shows reactive polypeptides of approximately 70 and 85 to 90 kd in addition to troponin T in both treated and autopsy heart sections. Neither reactivity in immunohistochemistry nor additional reactive polypeptides in Western blot are detectable when calpain or transglutaminase is inhibited during exposure of sections to high calcium. Troponin T crosslinking occurs also in isolated myofibrils, which show staining with BN-59 at either sarcomeric A or I bands. Labeling with TdT-mediated dUTP nick and labeling (TUNEL) to demonstrate apoptosis reveals DNA fragmentation in BN-59-positive myocytes. Thus, troponin T cross-linking occurs in human cardiac myocytes concomitantly with apoptosis and autopsy autolysis, suggesting that similar cytosolic alterations can be produced by different types of myocyte death.

Topics: Acrylamide; Acrylamides; Adolescent; Adult; Apoptosis; Blotting, Western; Calcium; Calpain; Child; Dipeptides; Female; Heart; Humans; Immunohistochemistry; Male; Middle Aged; Myocardium; Troponin; Troponin T

Cerebral deposition of amyloid beta-protein (Abeta) as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Abeta falls into two major subspecies defined by their C-termini, Abeta40 and Abeta42, ending in Val-40 and Ala-42, respectively. Although Abeta42 accounts for only approximately 10% of secreted Abeta, Abeta42 is the predominant species accumulated in senile plaques in AD brain and appears to be the initially deposited species. Its secretion level has recently been reported to be increased in the plasma or culture media of fibroblasts from patients affected by any of early-onset familial AD (FAD). Thus, inhibition of Abeta42 production would be one of the therapeutic targets for AD. However, there is little information about the cleavage mechanism via which Abeta40 and Abeta42 are generated and its relationship to intracellular protease activity. Here, we examined by well-characterized enzyme immunoassay the effects of calpain and proteasome inhibitors on the levels of Abeta40 and Abeta42 secretion by cultured cells. A calpastatin peptide homologous to the inhibitory domain of calpastatin, an endogenous calpain specific inhibitor, induced a specific increase in secreted Abeta42 relative to the total secreted Abeta level, a characteristic of the cultured cells transfected with FAD-linked mutated genes, while a proteasome specific inhibitor, lactacystin, showed no such effect. These findings suggest that the Abeta42 secretion ratio is modulated by the calpain-calpastatin system and may point to the possibility of exploring particular compounds that inhibit Abeta42 secretion through this pathway.

Topics: Acetylcysteine; Alzheimer Disease; Amyloid beta-Peptides; Calcimycin; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Embryo, Mammalian; Humans; Kidney; Peptide Fragments; Transfection

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

Topics: Antibody Specificity; Blotting, Western; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cells, Cultured; Chromatography, DEAE-Cellulose; Cytoskeletal Proteins; Dipeptides; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Extracellular Space; Humans; Hydrolysis; RNA, Messenger; Umbilical Veins

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm2 was applied to platelets, activation of mu-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm2, mu-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm2, applied for 5 min, caused mu-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both mu-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization.

Topics: Calpain; Dipeptides; Enzyme Inhibitors; Humans; Platelet Activation; Platelet Aggregation; Rheology; Stress, Mechanical; Talin; Time Factors

The intracellular thiol protease mu-calpain exists as a heterodimeric proenzyme, consisting of a large 80-kDa catalytic subunit and a smaller 30-kDa regulatory subunit. Activation of mu-calpain requires calcium influx across the plasma membrane and the subsequent autoproteolytic conversion of the 80-kDa large subunit to a 78-kDa "intermediate" and a 76-kDa fully autolyzed form. Currently, there is limited information on the substrate specificities and functional roles of these distinct active forms of mu-calpain within the cell. Using antibodies that can distinguish among the 80-, 78-, and 76-kDa forms of mu-calpain, we have demonstrated a close correlation between the autolytic generation of the 78-kDa enzyme and the proteolysis of the non-receptor tyrosine phosphatase, PTP-1B, in ionophore A23187-stimulated platelets. Time course studies revealed that pp60(c-)src proteolysis lagged well behind that of PTP-1B and correlated closely with the generation of the fully proteolyzed form of mu-calpain (76 kDa). In vitro proteolysis experiments with purified mu-calpain and immunoprecipitated PTP-1B or pp60(c-)src confirmed selective proteolysis of pp60(c-)src by the 76-kDa enzyme, whereas PTP-1B cleavage was mediated by both the 76- and 78-kDa forms of mu-calpain. Studies using selective pharmacological inhibitors against the different autolytic forms of mu-calpain have demonstrated that the initial conversion of the mu-calpain large subunit to the 78-kDa form is responsible for the reduction in platelet-mediated clot retraction, whereas complete proteolytic activation of mu-calpain (76 kDa) is responsible for the shedding of procoagulant-rich membrane vesicles from the cell surface. These studies demonstrate the existence of multiple active forms of mu-calpain within the cell, that have unique substrate specificities and distinct functional roles.

Topics: Antibodies; Blood Platelets; Calcimycin; Calpain; Cysteine Proteinase Inhibitors; Dimerization; Dipeptides; Egtazic Acid; Enzyme Activation; Enzyme Precursors; Humans; Kinetics; Macromolecular Substances; Molecular Weight; Organelles; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins pp60(c-src); Subcellular Fractions; Substrate Specificity

Two dipeptide aldehyde cell permeable calpain inhibitors, cBz-Val-Phe and calpeptin, have been assessed for their ability to prevent cytoskeletal proteolysis and loss of transparency in whole rat lenses. Calcium overload, induced by ionomycin in artificial aqueous humor with 1mM calcium, resulted in lens opacification and degradation of cytoskeletal proteins including spectrin, filensin, and vimentin. No such changes resulted from incubation in ionomycin in the absence of calcium. In calcium overload lenses both inhibitors gave some protection against cytoskeletal protein degradation and loss of transparency. These experiments indicate that calpain has a role in cortical opacification in high calcium lenses and that cell penetrating calpain inhibitors do indeed enter lens cells and reduce both proteolysis and opacification.

Topics: Animals; Calcium; Calpain; Cataract; Cattle; Cell-Free System; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Eye Proteins; Intermediate Filament Proteins; Ionophores; Lens, Crystalline; Light; Rats; Rats, Wistar; Scattering, Radiation; Spectrin; Vimentin

Using red cells as an experimental model, we previously showed that a limited degradation of certain membrane proteins by calpain (Ca2+-activated thiol protease) was a necessary prerequisite for cell fusion and that fusibility depended on the ratio of calpain to its endogenous inhibitor calpastatin. Here we show that fusion of rat L8 line myoblasts is accompanied by a dramatic change in the calpain/calpastatin ratio. The protein levels of mu-calpain and m-calpain increased only slightly during myoblast differentiation. In contrast, calpastatin diminished by a factor of 10 at the stages of myoblast alignment and start of fusion, allowing calpain activity to become apparent. Calpastatin reappeared at a late stage of myoblast fusion (myotube formation). The results indicate that calpastatin is regulated during myoblast differentiation, and that its diminution is important in determining the activity of the calpain required for myoblast fusion.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Fusion; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Kinetics; Molecular Weight; Muscle Fibers, Skeletal; Rats

Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp 125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.

Topics: Blood Platelets; Calcimycin; Calpain; Cell Adhesion Molecules; Cysteine Proteinase Inhibitors; Cytoskeleton; Dipeptides; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glycoproteins; Humans; Immunoblotting; Ionophores; Molecular Weight; Peptide Fragments; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Subcellular Fractions; Thrombin

The mechanism of plasma membrane blebbing (dissociation of the lipid bilayer from the membrane cytoskeleton) in hepatocyte injury is not known. The aim of this study was to investigate the role of calpain, a calcium-dependent cytosolic protease, in bleb formation induced by oxidative stress.. Hepatocytes from Wistar rats were injured with tertbutyl hydroperoxide in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a specific calpain inhibitor, calpeptin (Z-Leu-nLeu-H). Bleb formation was examined by phase-contrast and transmission electron microscopies. Intracellular calcium concentration was measured using Fura-2. Western blot analyses were performed for cytoskeletal proteins (talin, alpha-actinin, and vinculin) and the intermediate (activated) and proactivated forms of calpain mu.. tert-Butyl hydroperoxide induced a sustained increase in intracellular calciu, bleb formation, and, ultimately, hepatocyte death. Talin and alpha-actinin were degraded in a time-dependent manner, although no apparent changes of actin filament were observed. Before the cytoskeletal protein degradation, the intermediate form of calpain mu appeared as its proactivated form decreased. In addition, calpeptin or EGTA inhibited not only calpain mu activation but also cytoskeletal protein degradation and bleb formation.. In tert-butyl hydroperoxide-treated hepatocytes, the activation of calpain promotes membrane blebbing via degradation of cytoskeletal proteins.

Topics: Animals; Calcium; Calpain; Cell Membrane; Cytoskeletal Proteins; Dipeptides; Liver; Male; Microscopy, Electron; Peroxides; Rats; Rats, Wistar; Reactive Oxygen Species; tert-Butylhydroperoxide

The possible role of calpains in protein-tyrosine phosphorylation in platelets was examined by the use of the cell-permeant calpain inhibitor calpeptin. In platelets stimulated by 1 U/mL thrombin, protein-tyrosine phosphorylation was maximal after 2 minutes and was followed by protein-tyrosine dephosphorylation. Calpeptin (30 mumol/L) or vanadate (2 mmol/L) enhanced protein-tyrosine phosphorylation and delayed protein-tyrosine dephosphorylation. The effects of these two compounds were not additive. We also observed proteolysis of pp60src and autoproteolysis of mu-calpain. Cleavage of the former was significantly slower than that of the latter and slower than protein-tyrosine dephosphorylation. The activity of protein-tyrosine phosphatase in the platelet lysate was transiently increased to 190% by addition of Ca2+. Ca(2+)-dependent activation of protein-tyrosine phosphatase was not observed in the presence of leupeptin. Those observations suggest that platelet calpains may be involved in modulation of protein-tyrosine phosphorylation through activation of protein-tyrosine phosphatase rather than through the inactivation of pp60src, a mechanism that was previously suggested.

Topics: Blood Platelets; Calpain; Dipeptides; Humans; Phosphorylation; Protein Tyrosine Phosphatases; Vanadates

It has already been reported that, in vitro, intermediate filaments such as desmin and vimentin are very susceptible to proteolysis by calpains (Ca(2+)-activated cysteine proteinases). On the other hand, desmin and m-calpain are both present at the onset of myoblast fusion and throughout this phenomenon. Based on these observations, the aim of this study was to demonstrate, with cultured rat myoblasts, that the amount of desmin decreased significantly as multinucleated myotubes were formed. Using immunoblot analysis, it has been shown that the desmin concentration decreased 41% as myoblasts fuse. Moreover, under conditions which stimulate myoblast fusion, desmin concentration was reduced by 21% compared to the control culture. Under our experimental conditions, which lead to a reduced desmin level, the amount of m-calpain was increased about three-fold. These results suggested that m-calpain could be involved in myoblast fusion via desmin cleavage. This hypothesis was confirmed by the results obtained after calpeptin treatment. In the presence of this cell-penetrating inhibitor of calpains, desmin seems not to be degraded. Taking into account the observations obtained after different hydrolysis assays and as compared to those observed on cultured cells, it seems conceivable that m-calpain would be able to initiate desmin cleavage leading to the formation of proteolytic fragments which should be immediately degraded.

Topics: Animals; Calcium; Calpain; Cell Fusion; Cells, Cultured; Desmin; Dipeptides; Intermediate Filaments; Muscle Proteins; Muscle, Skeletal; Rats; Rats, Wistar

Calpain activity of nerve growth factor (NGF)-induced rat pheochromocytoma (PC12) cells shows a transient diminution in the early phase of differentiation. Calpain activity can be further decreased by a cell-permeable calpain inhibitor, calpeptin, which enhances the effect of NGF by stimulating neurite elongation. The number of neurites sprouted by one cell is not increased by calpeptin. A possible role of calpain inhibition during PC12 cells' early differentiation is discussed.

Topics: Animals; Calpain; Cell Differentiation; Dipeptides; Nerve Growth Factors; Neurites; PC12 Cells; Rats

Myonephropathic metabolic syndrome (MNMS) is a serious muscle reperfusion injury associated with acute renal failure. The exact pathogenesis of MNMS has not been fully elucidated, nor effective treatment, through the renal failure is thought to be a consequence of rhabdomyolysis. In the present study, the possible involvement of calpain in the lysis was investigated in a MNMS animal model employing a cell permeable calpain antagonist calpeptin. Male rabbits were subjected to bilateral hind leg ischaemia for 5 hours by clamping the distal aorta, followed by reperfusion for 3 hours. Blood pressure, plasma N-acethyl-beta-D-glucosaminidase (NAG) and the presence of myoglobinuria were serially determined. Blood pressure remained constant during the ischemic period but dropped by about 25% immediately after reperfusion. This was significantly attenuated by intraaortic administration of calpeptin. NAG gradually increased during ischemia and during reperfusion and this was also significantly reduced by calpeptin. Myoglobinuria appeared immediately after reperfusion, and was also attenuated by calpeptin. Calpeptin prevented lytic and degenerative changes of the hind leg muscles, determined by light and electron microscopy. Thus it is concluded that activation of calpain in skeletal muscle is an important etiologic factor of MNMS and that the occurrence of MNMS may be prevented by administration of a calpain antagonist.

Topics: Acetylglucosaminidase; Acute Kidney Injury; Animals; Calpain; Dipeptides; Hindlimb; Male; Muscles; Rabbits; Reperfusion Injury; Rhabdomyolysis; Syndrome

Platelets were incubated in the presence of calpeptin to inhibit calpain-mediated cytoskeleton proteolysis during further activation by Ca2+ ionophore A23187. The appearance of filamin and myosin subfragments (93 kDa and 135 kDa, respectively) was inhibited by low calpeptin doses (1 microgram/ml). Higher doses (10-20 micrograms/ml) were required to completely inhibit talin and filamin degradation. Vesiculation strongly depended on cytoskeleton proteolysis and was reduced by 60% when platelets were preincubated with 10 micrograms/ml calpeptin. Activated platelets bore longer and more filopods when pretreated with calpeptin. Filopods were straight and regular when high calpeptin doses were used, whereas they were shorter and broader with bloated surfaces when calpeptin was omitted. Some bloated areas were also found in straight filopods. These results suggest that the cytoskeleton proteolysis, and more specifically filamin proteolysis, induced bloating of filopod surfaces, thus facilitating fragmentation of filopod into vesicles.

Topics: Blood Platelets; Calcimycin; Calpain; Contractile Proteins; Cytoskeleton; Dipeptides; Filamins; Humans; Microfilament Proteins; Platelet Activation

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

The mechanisms of hepatocyte injury caused by exogenous superoxide were investigated with the use of cultured rat hepatocytes. Cell viability, cytosolic free calcium concentration and cell surface structure were observed. Superoxide was produced by adding hypoxanthine and xanthine oxidase to the buffer. Cytosolic free calcium concentration was calculated by means of ratio imaging of fura 2 fluorescence with multiparameter digitized microscopy. In the buffer containing 1.27 mmol/L of calcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the addition of hypoxanthine and xanthine oxidase, small blebs were recognized on the cell surface with a scanning electron microscope; then a gradual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentration had risen to about 700 nmol/L. Depriving the buffer of calcium (< 10 mumol/L) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the basal level (200 nmol/L). When ethylene glycol-bis (beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free calcium concentration decreased. Superoxide dismutase combined with catalase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpeptin, a Ca(2+)-dependent neutral protease inhibitor, did not affect the rise in cytosolic free calcium concentration but prevented cell injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Calpain; Catalase; Cell Death; Cell Survival; Cells, Cultured; Cytosol; Dipeptides; L-Lactate Dehydrogenase; Liver; Male; Microscopy, Electron, Scanning; Microscopy, Phase-Contrast; Nifedipine; Rats; Rats, Wistar; Superoxide Dismutase; Superoxides

Calpain is distributed ubiquitously in virtually every tissue (Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813-846), but its physiological role remains to be determined. The identification of its natural endogenous substrates would be of great interest. Since pp60src, a major tyrosine kinase in platelets, is known to be easily cleaved during purification from cells (Feder, D., and Bishop, J. M. (1990) J. Biol. Chem. 265, 8205-8211), we examined the possibility that it is an endogenous substrate of calpain. In the whole cell lysate from resting platelets, which was analyzed by Western blotting with monoclonal antibody 327, we found pp60src almost exclusively in a 60-kDa form, with a trace of 52-kDa form. Addition of A23187 (a calcium ionophore) or dibucaine, which are known to be activators of platelet calpain (Croall and DeMartino, 1991; Fox, J. E., Reynolds, C., Morrow, J. S., and Phillips, D. R. (1987) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990b) J. Cell Biol. 111, 483-493), caused dose- and time-dependent cleavage of actin-binding protein and p235 protein (talin). At the same time, loss of the 60-kDa species of pp60src and generation of the 52-kDa (occasionally seen as doublets) and 47-kDa species were detected by the Western blotting. In platelets aggregated by 1 unit/ml thrombin, apparently identical cleavage products were found. The cleavage of pp60src was inhibited by calpeptin (20 microM), an inhibitor of calpain (Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201-1208; Tsujinaka, T., Ariyoshi, H., Uemura, Y., Sakon, M., Kambayashi, J., and Mori, T. (1990) Life Sci. 46, 1059-1066; Fox, J. E., Clifford, C. C., and Austin, C. D. (1990) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990) J. Cell. Biol. 111, 483-493; Fox, J. E., Austin, C. D., Clifford, C. C., and Steffen, P. K. (1991) J. Biol. Chem. 266, 13289-13295). Addition of EGTA (3 mM) to the extracellular media completely inhibited the cleavage of actin-binding protein, talin, and pp60src in response to A23187 (1 microM). Intact pp60src was distributed in both cytosolic and particulate (membrane) fractions. Cleaved species were found exclusively in the cytosolic fraction. pp60src-associated enolase kinase activity was reduced. Thus, pp60src is an endogenous substrate for calpain, the cleavage of which may h

Topics: Antibodies, Monoclonal; Blood Platelets; Blotting, Western; Calcimycin; Calcium; Calpain; Dimethyl Sulfoxide; Dipeptides; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Humans; Microfilament Proteins; Molecular Weight; Platelet Aggregation; Proto-Oncogene Proteins pp60(c-src); Substrate Specificity; Talin

The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.

Topics: Blood Coagulation; Blood Platelets; Calcimycin; Calpain; Cell Membrane; Collagen; Dipeptides; Flow Cytometry; Humans; Microscopy, Electron, Scanning; Platelet Adhesiveness; Platelet Membrane Glycoproteins; Thrombin; Thromboplastin; von Willebrand Factor

Platelets activated by various agonists produce formation of vesicles shed from the plasma membrane (microparticles). However, the mechanism of microparticle (MP) formation has not been clarified yet. The aim of the present study was to determine the possibility of involvement of calpain (a Ca(2+)-dependent thiol protease) in MP formation. Washed platelets preincubated with calpeptin, a cell permeable calpain specific inhibitor, or with a vehicle were activated by thrombin plus collagen or by calcium ionophore A23187. Flow cytometry was used to detect the amount of microparticle formation by using murine monoclonal antibodies against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate labeled goat anti-mouse IgG. MP formation stimulated either by thrombin plus collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A23187 reached a plateau in approximately 5 min after activation, whereas that from platelets activated by thrombin plus collagen reached a plateau at 30 min following the stimulation. These time sequences corresponded well with those of degradation of actin-binding protein (ABP), a well known substrate of calpain, of platelets activated by these two stimulations. However, the inhibition of MP formation by calpeptin was more marked in the early stage (within 10 min) than in the late stage (after 30 min) of platelet activation. At 30 min after platelet activation by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 microM calpeptin, which inhibited hydrolysis of ABP almost completely. Our data suggest the involvement of calpain in the early stage (especially within 10 min) of microparticle formation.

Topics: Antigens, Human Platelet; Blood Platelets; Calcimycin; Calcium; Calpain; Cell Membrane; Collagen; Dipeptides; Enzyme Activation; Flow Cytometry; Humans; Hydrolysis; Microfilament Proteins; Platelet Activation; Talin; Thrombin

Ca(2+)-induced degradation of the neuronal inositol (1,4,5)-trisphosphate receptor, a protein which regulates Ca(2+)-release from intracellular stores, has been examined. The IP3-receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca(2+)-activated neutral protease (calpain). Incubation of membranes or immunopurified IP3-receptor with Ca2+ and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca(2+)-channel-containing part of the protein. These data suggest that activation of the IP3-receptor, by causing increases in intracellular [Ca2+], might result in degradation of the N-terminal, IP3-binding part of the receptor.

Topics: Animals; Astrocytes; Calcium; Calcium Channels; Calpain; Cell Membrane; Cerebellum; Chromatography, Affinity; Dipeptides; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leupeptins; Male; Molecular Weight; Neurons; Phosphorylation; Rats; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Testis; Tumor Cells, Cultured; Vas Deferens

To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca2+ ([Ca2+]i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 microM) nor EST (250 microM) altered the increase of [Ca2+]i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca2+]i back towards the basal level. These observations suggested that calpain might be involved in Ca2+ restoration. Then, the activity of Ca(2+)-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca(2+)-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca(2+)-ATPase activation induced by thrombin in a dose related manner. Ca(2+)-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca(2+)-ATPase activity was investigated. With a short incubation (8-15 s), Ca(2+)-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca2+ uptake was also increased by about 2-fold with a short incubation (8-15 s). For molecular characterization of the Ca(2+)-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca(2+)-ATPase was converted by calpain to a fragment of approximately 50 kD.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aspirin; Blood Platelets; Calcimycin; Calcium; Calcium-Transporting ATPases; Calpain; Cell Compartmentation; Dipeptides; Enzyme Activation; Homeostasis; Humans; Intracellular Membranes; Leucine; Multienzyme Complexes; Platelet Activation; Stimulation, Chemical; Thrombin

Smooth muscle proliferation is a prominent feature of the vascular response to mechanical injury. Accordingly, modulation of proliferation has important therapeutic implications for angioplasty restenosis. We have identified a subclass of thiol protease inhibitors (TPIs) that reversibly inhibit bovine aortic smooth muscle cell (BASMC) proliferation in vitro. To define the nature of this inhibition, an evaluation of selected steps in the cell cycle was undertaken. Treatment of BASMCs with benzyloxycarbonyl-Leu-norleucinal (calpeptin) at 100 microM and acetyl-Leu-Leu-norleucinal (TPI-1) at 50 microM was shown to cause a block of platelet-derived growth factor-BB as well as serum-inducible cell cycle progression at a point before the G1-S boundary, reducing the percentage of bromodeoxyuridine-positive cells from 87% to 5% over a 24-hour labeling period. Addition of TPI-1 at various times after serum addition to serum-deprived BASMCs showed 80% of the maximal block of DNA synthesis even when added 6 hours after serum. The cell cycle progression block was gradually lost as the delay from serum to TPI-1 application was increased from 6 to 12 hours. By Northern analysis of mRNA after serum addition, TPI-1 caused a fourfold decrease in the transient elevation of fos and myc proto-oncogene as well as a decrease in the levels of both muscle and nonmuscle actin mRNA induced early after serum addition. Flow cytometric analysis of DNA content and synthesis in BASMCs treated with TPI-1 or calpeptin additionally revealed the presence of a distinct cell cycle block in the G2-M compartment. In the aggregate, these results suggest the existence of more than one molecular site potentially involved in inhibition by TPI of cell cycling in BASMCs.

Topics: Animals; Blotting, Northern; Calpain; Cattle; Cell Cycle; Cell Division; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; DNA; Muscle, Smooth, Vascular

Benzyloxycarbonyl(Z)-Leu-nLeu-H (calpeptin) and Z-Leu-Met-H, cell-penetrating inhibitors of calpain, were found to block myoblast fusion without any effect on cell proliferation and alignment along their bipolar axis. They also inhibited the accumulation of creatine kinase during myogenesis. These effects were dose-dependent, and could be reversed upon removal of the drug from the culture medium. Furthermore, treatment of the inhibitors prevented the hydrolysis of filamin, which is sensitive to cleavage by calpain in vitro and interferes with actin-myosin filament formation by cross-linking F-actin molecules. On the other hand, leupeptin, which can also inhibit calpain in vitro but can not penetrate into cells, showed little or no effect on both myoblast fusion and filamin clevage. These results suggest that calpain may play an important role in cytoskeletal reorganization that is requisite for myoblast fusion. The role of calpain on the expression of muscle-specific proteins remains unknown.

Topics: Animals; Calpain; Cell Membrane Permeability; Cells, Cultured; Chick Embryo; Contractile Proteins; Dipeptides; Filamins; Membrane Fusion; Microfilament Proteins; Muscles

Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Basilar Artery; Calpain; Dipeptides; Dogs; Ischemic Attack, Transient; Isoquinolines; Naphthalenes; Piperazines; Polycyclic Compounds; Protein Kinase C; Vasoconstriction

The binding of calpain-I (Ca2+ activated neutral protease with high Ca2+ sensitivity) to the membranes of human platelets and the subsequent autolytic activation of calpain-I were analyzed by an immunoblot technique. In A23187 stimulated platelets, cytosolic calpain-I translocated to the membranes with autolysis in a Ca2+ dependent manner and simultaneously underwent autolysis. Although calpeptin, a cell permeable calpain inhibitor, inhibited autolysis of calpain-I, it was unable to prevent the translocation of calpain-I. In a cell re-constituted system, the membrane binding of calpain-I was also Ca2+ dependent and was significantly inhibited by a substrate of calpain. It was suggested that the binding of calpain-I to the membranes required the substrate binding site of this enzyme.

Topics: Biological Transport; Blood Platelets; Calcium; Calpain; Cell Membrane; Cytosol; Dipeptides; Enzyme Activation; Humans; Immunoblotting; Protein Binding

A calpain 1-protein kinase C (PKC) complex was isolated from rabbit skeletal muscle by hydrophobic interaction chromatography on phenyl-sepharose and by strong anion exchange chromatography on Q-Sepharose. Calpain 1 and kinase activities were then dissociated on a phenyl-Sepharose matrix using gradients of decreasing ionic strength. The purified PKC obtained corresponded to conventional PKC and was recognized by a monoclonal antibody specific for alpha and beta isotypes. Leupeptin, calpain inhibitor II, and the more selective calpain inhibitors calpeptin and MDL 28170 did not block the activation of the purified PKC by Ca2+ and phosphatidylserine.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Calpain; Dipeptides; Enzyme Activation; Enzyme Stability; Leupeptins; Male; Molecular Sequence Data; Muscles; Protein Kinase C; Rabbits

Calpeptin (a cell permeable synthetic peptide calpain inhibitor) inhibited the generation of thromboxane B2 (TxB2) by the direct inhibition on Tx synthetase in platelets at the concentrations more than 30 microM. Calpeptin, its analogues and E-64d (EST) were further examined with regard to cell permiability and inhibitory spectra. Among all compounds, only calpeptin inhibited the degradation of substrate proteins of calpain with negligible effect on TxB2 generation in intact platelets at the concentrations less than 30 microM. These concentrations of calpeptin did not inhibit the platelet aggregation, the elevation of [Ca2+], nor the formation of inositol 1,4,5-trisphosphate (IP3) in thrombin or collagen activated platelets. These results indicate that calpain dose not participate in the process of platelet activation induced by thrombin or collagen.

Topics: Arachidonic Acids; Calcium; Calpain; Collagen; Cyclooxygenase Inhibitors; Dipeptides; Humans; Inositol 1,4,5-Trisphosphate; Molecular Structure; Platelet Activation; Structure-Activity Relationship; Thrombin; Thromboxane B2; Thromboxane-A Synthase

N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor.

Topics: Animals; Blood Platelets; Calcimycin; Calpain; Cell Membrane Permeability; Dipeptides; Leupeptins; Microfilament Proteins; Papain; Peptides; Swine