calpain and Hypoxia

Topics: Animals; Calcium; Calpain; Caspases; Cysteine Endopeptidases; Hypoxia; Ischemia; Kidney Diseases; Kidney Tubules

Increasing evidence now suggests that excessive activation of calcium-dependent neutral proteases, calpains, could play a key or contributory role in the pathology of cerebral ischemia. This assumption has been supported in part by the suppressive or neuroprotective effects of calpain inhibitors on post-ischemic damage. Targeting calcium-activated proteolysis could be therefore an alternative strategy for protecting neurons against post-ischemic injury. The data of this review indicate that unregulated activation of calcium-dependent proteolysis plays a significant role in the brain damage that occurs following an ischemic insult and that selective and permanent calpain inhibitors may provide a powerfully effective therapeutic means of limiting neuronal damage.

Topics: Brain Ischemia; Calpain; Dipeptides; Humans; Hypoxia; Nerve Degeneration

Hypoxia-induced muscle wasting has been observed in several environmental and pathological conditions. However, the molecular mechanisms behind this loss of muscle mass are far from being completely elucidated, certainly in vivo. When studying the regulation of muscle mass by environmental hypoxia, many confounding factors have to be taken into account, such as decreased protein ingestion, sleep deprivation or reduced physical activity, which make difficult to know whether hypoxia per se causes a reduction in muscle mass.. We hypothesized that acute exposure to normobaric hypoxia (11% O2 ) would repress the activation of the mTOR pathway usually observed after a meal and would activate the proteolytic pathways in skeletal muscle.. Fifteen subjects were exposed passively for 4 h to normoxic and hypoxic conditions in a random order after consumption of a light breakfast. A muscle biopsy and a blood sample were taken before, after 1 and 4 h of exposure.. After 4 h, plasma insulin concentration and the phosphorylation state of PKB and S6K1 in skeletal muscle were higher in hypoxia than in normoxia (P < 0.05). At the same time, Redd1 mRNA level was upregulated (P < 0.05), whilst MAFbx mRNA decreased (P < 0.05) in hypoxia compared with normoxia. Proteasome, cathepsin L and calpain activities were not altered by environmental hypoxia.. Contrary to our hypothesis and despite an increase in the mRNA level of Redd1, an inhibitor of the mTORC1 pathway, short-term acute environmental hypoxia induced a higher response of PKB and S6K1 to a meal, which may be due to increased plasma insulin concentration.

Topics: Acute Disease; Atmosphere Exposure Chambers; Blood Glucose; Breakfast; Calpain; Cathepsin L; Cross-Over Studies; Humans; Hydrocortisone; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Male; Muscle Proteins; Muscle, Skeletal; Oximetry; Oxygen; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; SKP Cullin F-Box Protein Ligases; TOR Serine-Threonine Kinases; Transcription Factors; Young Adult

Hypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types. In epithelial cancer, hypoxia induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective intervention to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as a key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low extracellular matrix (ECM)-adhesive, predominantly bleb-based amoeboid movement, which was maintained by a low-oxidative and -glycolytic energy metabolism ("eco-mode"). Hypoxia induced calpain-2-mediated amoeboid conversion by deactivating β1 integrins through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation or pharmacological inhibition of calpain-2 restored talin-1 integrity and β1 integrin engagement and reverted amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced amoeboid conversion in the orthotopic mouse dermis and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/β1 integrin axis as a druggable mechanosignaling program that conserves energy yet enables metastatic dissemination that can be reverted by interfering with calpain activity.

Topics: Animals; Calpain; Cell Line, Tumor; Cell Movement; Head and Neck Neoplasms; Humans; Hypoxia; Integrin beta1; Mice; Neoplasm Metastasis; Talin

Calpain-1, a calcium-activated neutral cysteine proteases, has been reported to be involved in the formation of pulmonary hypertension. HIF-1α, an oxygen-sensitive transcription factor, has been reported to activate genes involved in cell proliferation and extracellular matrix recombination. This study was designed to investigate the effect of calpain-1 in hypoxic pulmonary hypertension (HPH) and to explore whether there is a relationship between calpain-1 and HIF-1α in this disease. In the hypoxia-induced model of HPH, we found that hypoxia resulted in increased right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodelling and collagen deposition in lung tissues of mice. The levels of calpain-1 and HIF-1α were up-regulated in the lung tissues of hypoxia-treated mice and pulmonary arterial smooth muscle cells (PASMCs). Knock-out of calpain-1 restrained haemodynamic and histological changes induced by chronic hypoxia in mice, and inhibition of calpain-1 also repressed the abnormal proliferation and migration of PASMCs. Besides, knock-out or inhibition of calpain-1 suppressed hypoxia-induced expression of HIF-1α, VEGF, PCNA, TGF-β1, MMP2 and collagen I in vivo and in vitro. While inhibition of HIF-1α abolished the above effects of calpain-1. Furthermore, we found that calpain-1 mediates the expression of HIF-1α through NF-κB (P65) under hypoxia conditions. In conclusion, our results suggest that calpain-1 plays a pivotal role in hypoxia-induced pulmonary vascular remodelling and fibrosis through HIF-1α, providing a better understanding of the pathogenesis of HPH.

Topics: Animals; Calpain; Cell Proliferation; Fibrosis; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Myocytes, Smooth Muscle; Pulmonary Artery; Vascular Remodeling

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodeling in pulmonary hypertension (PH). It has been reported that miR-137 inhibits the proliferation of tumor cells. However, whether miR-137 is involved in PH remains unclear. In this study, male Sprague-Dawley rats were subjected to 10% O

Topics: Animals; Calpain; Cell Hypoxia; Cell Movement; Cell Proliferation; Cells, Cultured; Hypertension, Pulmonary; Hypoxia; Male; MicroRNAs; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vascular Remodeling

Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber.. Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation.. Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945.. An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Calpain; Carbamates; Caspase 3; Cells, Cultured; Child; Cytosol; DNA Fragmentation; Enzyme Activation; Humans; Hypoxia; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Macaca mulatta; Middle Aged; Retinal Diseases; Retinal Ganglion Cells

Sarcomas are still unsolved therapeutic challenges. Cancer stem cells are believed to contribute to sarcoma development, but lack of specific markers prevents their characterization and targeting. Here, we show that calpain-6 expression is associated with cancer stem cell features. In mouse models of bone sarcoma, calpain-6-expressing cells have unique tumor-initiating and metastatic capacities. Calpain-6 levels are especially high in tumors that have been successfully propagated in mouse to establish patient-derived xenografts. We found that calpain-6 levels are increased by hypoxia in vitro and calpain-6 is detected within hypoxic areas in tumors. Furthermore, calpain-6 expression depends on the stem cell transcription network that involves Oct4, Nanog, and Sox2 and is activated by hypoxia. Calpain-6 knockdown blocks tumor development in mouse and induces depletion of the cancer stem cell population. Data from transcriptomic analyses reveal that calpain-6 expression in sarcomas inversely correlates with senescence markers. Calpain-6 knockdown suppresses hypoxia-dependent prevention of senescence entry and also promotion of autophagic flux. Together, our results demonstrate that calpain-6 identifies sarcoma cells with stem-like properties and is a mediator of hypoxia to prevent senescence, promote autophagy, and maintain the tumor-initiating cell population. These findings open what we believe is a novel therapeutic avenue for targeting sarcoma stem cells.

Topics: Animals; Autophagy; Biomarkers; Calpain; Carcinogenesis; Cell Line, Tumor; Cellular Senescence; Disease Models, Animal; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Hypoxia; Male; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Nanog Homeobox Protein; Neoplasms; Neoplastic Stem Cells; Octamer Transcription Factor-3; Sarcoma; SOXB1 Transcription Factors; Xenograft Model Antitumor Assays

High altitude associated hypobaric hypoxia is one of the cellular and environmental perturbation that alters proteostasis network and push the healthy cell towards loss of muscle mass. The present study has elucidated the robust proteostasis network and signaling mechanism for skeletal muscle atrophy under chronic hypobaric hypoxia (CHH).. Male Sprague Dawley rats were exposed to simulated hypoxia equivalent to a pressure of 282 torr for different durations (1, 3, 7 and 14 days). After CHH exposure, skeletal muscle tissue was excised from the hind limb of rats for biochemical analysis.. Chronic hypobaric hypoxia caused a substantial increase in protein oxidation and exhibited a greater activation of ER chaperones, glucose-regulated protein-78 (GRP-78) and protein disulphide isomerase (PDI) till 14d of CHH. Presence of oxidized proteins triggered the proteolytic systems, 20S proteasome and calpain pathway which were accompanied by a marked increase in [Ca2+]. Upregulated Akt pathway was observed upto 07d of CHH which was also linked with enhanced glycogen synthase kinase-3β (GSk-3β) expression, a negative regulator of Akt. Muscle-derived cytokines, tumor necrosis factor-α (TNF-α), interferon-ϒ (IFN-©) and interleukin-1β (IL-1β) levels significantly increased from 07d onwards. CHH exposure also upregulated the expression of nuclear factor kappa-B (NF-κB) and E3 ligase, muscle atrophy F-box-1 (Mafbx-1/Atrogin-1) and MuRF-1 (muscle ring finger-1) on 07d and 14d. Further, severe hypoxia also lead to increase expression of ER-associated degradation (ERAD) CHOP/ GADD153, Ub-proteasome and apoptosis pathway.. The disrupted proteostasis network was tightly coupled to degradative pathways, altered anabolic signaling, inflammation, and apoptosis under chronic hypoxia. Severe and prolonged hypoxia exposure affected the protein homeostasis which overwhelms the muscular system and tends towards skeletal muscle atrophy.

Topics: Animals; Apoptosis; Calcium; Calpain; Cytokines; Glycogen Synthase Kinase 3 beta; Heat-Shock Proteins; Hypoxia; Male; Muscle, Skeletal; Proteasome Endopeptidase Complex; Protein Carbonylation; Proteostasis; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ubiquitin-Protein Ligases; Up-Regulation

The present study aimed to investigate how bats protect their brain in a hypothermic situation. Formosan leaf-nosed bats (Hipposideros terasensis) were used in this study and treated under three conditions: room temperature (25±1°C), low temperature (4±1°C), and hibernation. The reactive oxygen species (ROS) levels in the blood and apoptosis-related proteins in the brain tissue were assessed and then compared among those bats under three conditions. Our results showed that the blood ROS levels of bats treated under conditions of low temperature and hibernation were significantly reduced compared with bats treated under the condition of room temperature. Both immunohistochemistry and immunoblotting expressions of hypoxia, inflammation, and apoptosis-related proteins in the brain tissue of bats treated under the condition of hibernation were significantly reduced compared with those bats treated under conditions of room temperature and low temperature. Thus, we suggested that bats can protect the brain in cold environment by reducing blood ROS levels and decreasing expressions of hypoxia, inflammation, and apoptosis-related proteins in the brain. Possible protection mechanisms involved in hypothermic adaptations need to be further clarified.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Calpain; Caspase 12; Caspase 3; Chiroptera; Cold Temperature; Echolocation; Genes, bcl-2; Hibernation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Inflammation; Neuroprotection; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (α

Topics: Anemia, Sickle Cell; Animals; Blood Coagulation; Blood Platelets; Calpain; Disease Models, Animal; Female; Humans; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Activation

Calpain mediates collagen synthesis and cell proliferation and plays an important role in pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). In the present study, we investigated whether and how calpain is activated by PAH mediators in pulmonary artery smooth muscle cells (PASMCs). These data show that smooth muscle-specific knockout of calpain attenuated and knockout of calpastatin potentiated pulmonary vascular remodeling and pulmonary hypertension. Treatment of PASMCs with the PAH mediators platelet-derived growth factor (PDGF), serotonin, H2O2, endothelin-1, and IL-6 caused significant increases in calpain activity, cell proliferation, and collagen-I protein level without changes in protein levels of calpain-1 and -2. The calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA/AM) did not affect calpain activation, but the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 and knocking down of calpain-2 prevented calpain activation in PAH mediator-treated PASMCs. Mass spectrometry data showed that the phosphorylation of calpain-2 at serine (Ser) 50 was increased and the phosphorylation of calpain-2 at Ser369 was decreased in PDGF-treated PASMCs. The PDGF-induced increase in Ser50 phosphorylation of calpain-2 was prevented by PD98059, whereas dephosphorylation of calpain-2 at Ser369 was blocked by the protein phosphatase 2A inhibitor fostriecin. Furthermore, smooth muscle of pulmonary arteries in PAH animal models and patients with PAH showed higher levels of phospho-Ser50-calpain-2 (P-Ser50) and lower levels of phospho-Ser369-calpain-2 (P-Ser369). These data support that calpain modulates pulmonary vascular remodeling in PAH. PAH mediator-induced activation of calpain is caused by ERK1/2-dependent phosphorylation of calpain-2 at Ser50 and protein phosphatase 2A-dependent dephosphorylation of calpain-2 at Ser369 in pulmonary vascular remodeling of PAH.

Topics: Animals; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Enzyme Activation; Enzyme Activators; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Hypertension, Pulmonary; Hypoxia; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Pulmonary Artery; RNA Interference; Signal Transduction; Transfection; Vascular Remodeling

Obesity is known to increase asthma risk and severity. Increased levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, are associated with mitochondrial toxicity, asthma, and metabolic syndrome. IL-4 upregulates the expression of protein arginine methyltransferases, which are essential for ADMA formation. Importantly, cross-talk between IL-4, ADMA, and mitochondrial dysfunction could explain how obesity and IL-4 can synergize to exacerbate allergic inflammation.. We sought to investigate how IL-4, a key asthma-associated cytokine, can influence ADMA-related effects on lungs.. BEAS2B (bronchial epithelial) cells were treated with IL-4 followed by ADMA and investigated for oxo-nitrative stress and resultant mitochondrial toxicity after 48 hours by using flow cytometry, confocal imaging, immunoblotting, and fluorimetric assays.. IL-4-induced mitotoxicity in BEAS2B cells was significantly higher in the presence of exogenous ADMA. IL-4 treatment led to proteolytic degradation of dimethylarginine dimethylaminohydrolase 2, which catabolizes ADMA. IL-4 pretreatment was associated with increased intracellular ADMA accumulation and increased ADMA-induced mitotoxicity. Airway epithelial cells treated with IL-4 followed by ADMA showed exaggerated oxo-nitrative stress and potent induction of the cellular hypoxic response, despite normoxic conditions. The hypoxic response was associated with reduced mitochondrial function but was reversible by overexpression of the mitochondrial biogenesis factor, mitochondrial transcription factor A.. We conclude that IL-4 promotes intracellular ADMA accumulation, leading to mitochondrial loss through oxo-nitrative stress and hypoxic response. This provides a novel understanding of how obesity, with high ADMA levels, and asthma, with high IL-4 levels, might potentiate each other and highlights the potential of mitochondrial-targeted therapeutics in obese subjects with asthma.

Topics: Amidohydrolases; Apoptosis; Arginine; Asthma; Calpain; Cell Line; Cells, Cultured; Cytokines; Epithelial Cells; Humans; Hypoxia; Inflammation Mediators; Interleukin-4; Mitochondria; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Proteolysis; Reactive Oxygen Species; Respiratory Mucosa

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Cell Movement; Cell Proliferation; Cytomegalovirus; Disease Progression; Extracellular Space; Heart Function Tests; Humans; Hypertension, Pulmonary; Hypoxia; Intracellular Space; Male; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Smooth Muscle; Promoter Regions, Genetic; Pulmonary Artery

Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism.

Topics: Animals; Becaplermin; Benzamides; Calpain; Cell Proliferation; Cells, Cultured; Collagen; Cysteine Proteinase Inhibitors; Dioxoles; Dipeptides; Disease Models, Animal; Enzyme Activation; Humans; Hypertension, Pulmonary; Hypoxia; Mechanistic Target of Rapamycin Complex 2; Mice, Knockout; Multiprotein Complexes; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-sis; Pulmonary Artery; Receptors, Transforming Growth Factor beta; Ribonucleosides; RNA Interference; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection; Transforming Growth Factor beta1; Vascular Remodeling

To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms.
. Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR.
. RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression.
. Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.. 目的：研究钙蛋白酶(calpain)在低氧诱导肺动脉高压肺血管重构中的作用及机制。方法：SD大鼠随机分为低氧模型组和常氧对照组。插管法测定大鼠右心室收缩压及平均肺动脉压，右心室/(左心室+室间隔)比值评价右心肥厚指数，HE染色检测血管重构情况，分别采用实时荧光定量PCR和Western印迹检测肺动脉calpain-1，-2和-4 mRNA和蛋白的表达。原代培养的大鼠肺动脉平滑肌细胞分为4组：常氧对照组、常氧对照+
MDL28170(calpain抑制剂)组、低氧模型组、低氧模型+MDL28170组。MTS及流式细胞术观察细胞增殖情况，实时荧光定量PCR检测Ki-67及增殖细胞核抗原(proliferating cell nuclear antigen，PCNA) mRNA表达情况。结果：与常氧对照组大鼠比较，低氧模型组大鼠右心室收缩压、平均肺动脉压及右心肥厚指数显著增加，肺动脉血管显著重构；同时，低氧模型组大鼠肺动脉中calpain-1，-2，-4 mRNA和蛋白表达也显著上调。与常氧对照组细胞比较，低氧模型组细胞显著增殖，MDL28170可显著抑制低氧诱导的肺动脉平滑肌细胞增殖，同时逆转低氧诱导的Ki-67和PCNA mRNA表达上调。结论：calpain介导低氧诱导的肺动脉高压肺血管重构，其机制可能与介导低氧诱导的肺动脉平滑肌细胞增殖有关。.

Topics: Animals; Calpain; Cell Proliferation; Dipeptides; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Ki-67 Antigen; Myocytes, Smooth Muscle; Proliferating Cell Nuclear Antigen; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Up-Regulation; Vascular Remodeling

The vascular ischemic hypothesis attributes nerve damage in the retina to decreased blood flow in the ophthalmic artery, reduced oxygenation, and impaired axonal transport. Activation of calpain enzymes contributes to retinal cell death during hypoxia. However, we still do not know in which specific retinal layers calpains are activated. Thus, the purpose of the present study was to investigate where and when calpains are activated in an improved culture model of hypoxic monkey retina.. Monkey retinal explants were cultured on microporous membranes with the retinal ganglion cell (RGC) side facing up. Explants were incubated under hypoxic conditions, with or without additional reoxygenation. When it was used, the calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting assays for α-spectrin, calpains 1 and 2, calpastatin, β-III tubulin, and γ-synuclein were performed with specific antibodies. Cell death was assessed by TUNEL staining.. Under normoxic conditions, TUNEL-positive cells were minimal in our improved culture conditions. As early as 8 hours after hypoxia, the 150-kDa calpain-specific α-spectrin breakdown product appeared in the nerve fiber layer (NFL), where calpains 1 and 2 were localized. TUNEL-positive RGCs then increased at later time periods. The calpain inhibitor SNJ-1945 ameliorated changes induced by hypoxia or hypoxia/reoxygenation.. During hypoxia/reoxygenation in an improved, relevant monkey model, calpains were first activated in the NFL, followed by death of the parent RGCs. This observation suggest that calpain-induced degeneration of retinal nerve fibers may be an underlying mechanism for RGC death in hypoxic retinal neuropathies.

Topics: Animals; Calpain; Cell Death; Cells, Cultured; Disease Models, Animal; Hypoxia; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Macaca mulatta; Nerve Fibers; Retinal Diseases; Retinal Ganglion Cells

Calpain proteases are known to be involved in retinal cell death in animal models. The purpose of the present study was to test for calpain activation in human retinas cultured under hypoxic conditions.. Calpain activation was detected by immunoblotting for calpain substrates in human and monkey retinas cultured in gas generating pouches to reduce oxygen.. Hypoxia caused activation of calpains as measured by accumulation of the calpain-specific 145 kDa α-spectrin breakdown product. Opsin-1 (photoreceptor marker) and vimentin (Müller cell marker) were degraded. Calpain inhibitor SNJ-1945 ameliorated these changes. Results were similar to comparative data from cultured monkey retinas.. In cultured human retina, hypoxia caused activation of calpain and subsequent proteolysis of critical substrates. The efficacy of SNJ-1945 in ameliorating these changes indicated that it might be useful to test as a drug for protecting against pathologic proteolysis of photoreceptor and Müller cells.

Topics: Aged; Aged, 80 and over; Animals; Calpain; Carbamates; Cell Death; Cells, Cultured; Disease Models, Animal; Female; Haplorhini; Humans; Hypoxia; Immunoblotting; Male; Middle Aged; Peptide Hydrolases; Proteolysis; Retina

Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca(2+) level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments.

Topics: Adult; Altitude Sickness; Animals; Blood Platelets; Calpain; Disease Models, Animal; Enzyme Activation; Humans; Hypoxia; Male; Platelet Activation; Proteome; Rats; Rats, Sprague-Dawley; Thrombophilia; Thrombosis

Cerebral ischemia is characterized by an early disruption of GABAergic neurotransmission contributing to an imbalance of the excitatory/inhibitory equilibrium and neuronal death, but the molecular mechanisms involved are not fully understood. Here we report a downregulation of GABA(A) receptor (GABA(A)R) expression, affecting both mRNA and protein levels of GABA(A)R subunits, in hippocampal neurons subjected to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Similar alterations in the abundance of GABA(A)R subunits were observed in in vivo brain ischemia. OGD reduced the interaction of surface GABA(A)R with the scaffold protein gephyrin, followed by clathrin-dependent receptor internalization. Internalization of GABA(A)R was dependent on glutamate receptor activation and mediated by dephosphorylation of the β3 subunit at serine 408/409. Expression of phospho-mimetic mutant GABA(A)R β3 subunits prevented receptor internalization and protected hippocampal neurons from ischemic cell death. The results show a key role for β3 GABA(A)R subunit dephosphorylation in the downregulation of GABAergic synaptic transmission in brain ischemia, contributing to neuronal death. GABA(A)R phosphorylation might be a therapeutic target to preserve synaptic inhibition in brain ischemia.

Topics: Animals; Calpain; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Glucose; Hippocampus; Humans; Hypoxia; Infarction, Middle Cerebral Artery; Neurons; Phosphorylation; Protein Subunits; Rats; Rats, Wistar; Receptors, GABA-B; Time Factors

Topics: Animals; Blood Platelets; Calpain; Humans; Hypoxia; Male; Thrombosis

Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke.

Topics: Animals; Calpain; Cell Death; Corpus Striatum; Cyclin-Dependent Kinase 5; Disease Models, Animal; Estrogens; Female; Glial Fibrillary Acidic Protein; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Male; Mice, Knockout; Nerve Tissue Proteins; Nervous System Diseases; Neurons; Phosphotransferases; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Time Factors; Tissue Plasminogen Activator

Previous studies reported that chronic intermittent hypoxia (CIH) results in an imbalanced expression of hypoxia-inducible factor-α (HIF-α) isoforms and oxidative stress in rodents, which may be due either to the direct effect of CIH or indirectly via hitherto uncharacterized mechanism(s). As neural activity is a potent regulator of gene transcription, we hypothesized that carotid body (CB) neural activity contributes to CIH-induced HIF-α isoform expression and oxidative stress in the chemoreflex pathway. Experiments were performed on adult rats exposed to CIH for 10 days. Rats exposed to CIH exhibited: increased HIF-1α and decreased HIF-2α expression; increased NADPH oxidase 2 and decreased superoxide dismutase 2 expression; and oxidative stress in the nucleus tractus solitarius and rostral ventrolateral medulla as well as in the adrenal medulla (AM), a major end organ of the sympathetic nervous system. Selective ablation of the CB abolished these effects. In the AM, sympathetic activation by the CB chemoreflex mediates CIH-induced HIF-α isoform imbalance via muscarinic acetylcholine receptor-mediated Ca(2+) influx, and the resultant activation of mammalian target of rapamycin pathway and calpain proteases. Rats exposed to CIH presented with hypertension, elevated sympathetic activity and increased circulating catecholamines. Selective ablation of either the CB (afferent pathway) or sympathetic innervation to the AM (efferent pathway) abolished these effects. These observations uncover CB neural activity-dependent regulation of HIF-α isoforms and the redox state by CIH in the central and peripheral nervous systems associated with the chemoreflex.

Topics: Adrenal Medulla; Animals; Basic Helix-Loop-Helix Transcription Factors; Calcium; Calpain; Carotid Body; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; NADPH Oxidases; Oxidative Stress; Protein Isoforms; Rats; Rats, Sprague-Dawley; Reflex; Solitary Nucleus; Superoxide Dismutase; TOR Serine-Threonine Kinases

Previous studies have demonstrated that endoplasmic reticulum (ER) stress is activated in Alzheimer's disease (AD) brains. ER stress-triggered unfolded protein response (UPR) leads to tau phosphorylation and neuronal death.. In this study, we tested the hypothesis that hypoxia-induced m-calpain activation is involved in ER stress-mediated AD pathogenesis.. We employed a hypoxic exposure in APP/PS1 transgenic mice and SH-SY5Y cells overexpressing human Swedish mutation APP (APPswe).. We observed that hypoxia impaired spatial learning and memory in the APP/PS1 mouse. In the transgenic mouse brain, hypoxia increased the UPR, upregulated apoptotic signaling, enhanced the activation of calpain and glycogen synthase kinase-3β (GSK3β), and increased tau hyperphosphorylation and β-amyloid deposition. In APPswe cells, m-calpain silencing reduced hypoxia-induced cellular dysfunction and resulted in suppression of GSK3β activation, ER stress and tau hyperphosphorylation reduction as well as caspase pathway suppression.. These findings demonstrate that hypoxia-induced abnormal calpain activation may increase ER stress-induced apoptosis in AD pathogenesis. In contrast, a reduction in the expression of the m-calpain isoform reduces ER stress-linked apoptosis that is triggered by hypoxia. These findings suggest that hypoxia-triggered m-calpain activation is involved in ER stress-mediated AD pathogenesis. m-calpain is a potential target for AD therapeutics.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Calpain; Cell Line, Tumor; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Female; Hippocampus; Humans; Hypoxia; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Presenilin-1; Random Allocation

Chronic hypoxia has been reported to contribute to the development of Alzheimer's disease (AD). However, the mechanism of hypoxia in the pathogenesis of AD remains unclear. The purpose of this study was to investigate the effects of chronic hypoxia treatment on β-amyloid, tau pathologies, and the behavioral consequences in the double transgenic (APP/PS1) mice. Double transgenic mice (APP/PS1 mice) were treated with hypoxia, and spatial learning and memory abilities of mice were assessed in the Morris water maze. β-amyloid level and plaque level in APP/PS1 double transgenic mice were detected by immunohistochemistry. Protein tau, p35/p25, cyclin-dependent kinase 5 (CDK5), and calpain were detected by western blotting analysis. Chronic hypoxia treatment decreased memory and cognitive function in AD mice. In addition, chronic hypoxia treatment resulted in increased senile plaques, accompanying with increased tau phosphorylation. The hypoxia-induced increase in the tau phosphorylation was associated with a significant increase in the production of p35 and p25 and upregulation of calpain, suggesting that hypoxia induced aberrant CDK5/p25 activation via upregulation of calpain. Our results showed that chronic hypoxia exposure accelerates not only amyloid pathology but also tau pathology via calpain-mediated tau hyperphosphorylation in an AD mouse model. These pathological changes possibly contribute to the hypoxia-induced behavioral change in AD mice.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Calpain; Cyclin-Dependent Kinase 5; Hypoxia; Maze Learning; Memory; Mice; Nerve Tissue Proteins; Phosphorylation; Phosphotransferases; tau Proteins; Up-Regulation

The most frequently reported symptom of exposure to high altitude is loss of body mass and decreased performance which has been attributed to altered protein metabolism affecting skeletal muscles mass. The present study explores the mechanism of chronic hypobaric hypoxia mediated skeletal muscle wasting by evaluating changes in protein turnover and various proteolytic pathways. Male Sprague-Dawley rats weighing about 200 g were exposed to hypobaric hypoxia (7,620 m) for different durations of exposure. Physical performance of rats was measured by treadmill running experiments. Protein synthesis, protein degradation rates were determined by (14)C-Leucine incorporation and tyrosine release, respectively. Chymotrypsin-like enzyme activity of the ubiquitin-proteasome pathway and calpains were studied fluorimetrically as well as using western blots. Declined physical performance by more than 20%, in terms of time taken in exhaustion on treadmill, following chronic hypobaric hypoxia was observed. Compared to 1.5-fold increase in protein synthesis, the increase in protein degradation was much higher (five-folds), which consequently resulted in skeletal muscle mass loss. Myofibrillar protein level declined from 46.79 ± 1.49 mg/g tissue at sea level to 37.36 ± 1.153 (P < 0.05) at high altitude. However, the reduction in sarcoplasmic proteins was less as compared to myofibrillar protein. Upregulation of Ub-proteasome pathway (five-fold over control) and calpains (three-fold) has been found to be important factors for the enhanced protein degradation rate. The study provided strong evidences suggesting that elevated protein turnover rate lead to skeletal muscle atrophy under chronic hypobaric hypoxia via ubiquitin-proteasome pathway and calpains.

Topics: Animals; Atrophy; Calpain; Carbon Radioisotopes; Hypoxia; Male; Motor Activity; Muscle, Skeletal; Myofibrils; Pressure; Proteasome Endopeptidase Complex; Proteins; Proteolysis; Rats; Rats, Sprague-Dawley; Ubiquitin

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.

Topics: Animals; Antioxidants; Calpain; Down-Regulation; Enzyme Activation; Hypoxia; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Myocardial Reperfusion Injury; Myocardium; Oxidants; Oxidative Stress; Perfusion; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Sarcolemma; Sodium-Potassium-Exchanging ATPase; Time Factors; Ventricular Function, Left; Ventricular Pressure

In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia. Mechanistically, these calpain inhibitors improved stability and organization of the actin cytoskeleton in retinal endothelial cells undergoing capillary morphogenesis in vitro, and they similarly improved organization of actin cables within new blood vessels in vivo. Hypoxia induced calpain activity in retinal endothelial cells and severely disrupted the actin cytoskeleton, whereas calpain inhibitors preserved actin cables under hypoxic conditions. Collectively, these findings support the hypothesis that hyper-activation of calpains by hypoxia contributes to disruption of the retinal endothelial cell cytoskeleton, resulting in formation of neovessels that are defective both architecturally and functionally. Modest suppression of calpain activity with calpain inhibitors restores cytoskeletal architecture and promotes formation of a functional neovasculature, thereby reducing underlying hypoxia. In sharp contrast to "anti-angiogenesis" strategies that cannot restore normoxia and may aggravate hypoxia, the therapeutic strategy described here does not inhibit neovascularization. Instead, by improving the function of neovascularization to reduce underlying hypoxia, moderate calpain inhibition offers a method for alleviating retinal ischemia, thereby suggesting a new treatment paradigm based on improvement rather than inhibition of new blood vessel growth.

Topics: Actins; Animals; Calpain; Catalytic Domain; Cell Line; Cysteine Proteinase Inhibitors; Cytoskeleton; Endothelial Cells; Glycoproteins; Humans; Hypoxia; Mice; Mice, Inbred C57BL; Retina; Retinal Diseases; Retinal Neovascularization; Retinal Vessels

This study was conducted to investigate the effect of flavonoids, a major family of antioxidants contained in foods, on retinal ganglion cell (RGC) death induced by hypoxia, excessive glutamate levels, and oxidative stress. Moreover, to assess the structure-activity relationships of flavonoids, three types of flavonoids with different numbers of hydroxyl groups and varieties of sugar chains were studied.. Three kinds of flavonoids-nicotiflorin, rutin, and quercitrin-were used. The death of neonatal rat purified RGCs was induced by hypoxic conditions (5% O(2), 5% CO(2), 37 °C) for 12 h, 25 µM glutamate over three days, or oxidative stress by depleting antioxidants from the medium for 24 h. RGC survival rates were calculated under each condition and compared with vehicle cultures. Modification of cell death signaling after stress-induced apoptosis and necrosis by flavonoids was assessed using caspase-3 and calpain immunoreactivity assays.. Under hypoxic and glutamate stress, both nicotiflorin and rutin significantly increased the RGC survival rate at 1 nM or higher, while quercitrin increased it at 100 nM or higher. Under oxidative stress, nicotiflorin, rutin, and quercitrin also significantly increased the RGC survival rate at 1 nM, 0.1 nM, and 100 nM or higher, respectively. Rutin significantly inhibited the induction of caspase-3 under both hypoxia and excessive glutamate stress, as well as blocking the induction of calpain during oxidative stress.. Nicotiflorin and rutin showed neuroprotective effects on hypoxia-, glutamate- or oxidative stress-induced RGC death at concentrations of 1 nM or higher. The presence of a specific sugar side chain (rutinoside) may enhance neuroprotective activity.

Topics: Animals; Antioxidants; Apoptosis; Calpain; Caspase 3; Caspase Inhibitors; Cell Culture Techniques; Cell Survival; Dose-Response Relationship, Drug; Flavonoids; Glutamic Acid; Hypoxia; Neuroprotective Agents; Oxidative Stress; Phenols; Quercetin; Rats; Rats, Wistar; Reactive Oxygen Species; Retinal Ganglion Cells; Rutin; Structure-Activity Relationship

Cell death occurring in human retina during AMD, high IOP, and diabetic retinopathy could be caused by activation of calpain or caspase proteolytic enzymes. The purpose of the present study was to determine whether calpains and/or caspase-3 were involved in cell death during retinal hypoxia in a monkey model.. Dissociated monkey retinal cells were cultured for two weeks and subjected to 24-hour hypoxia/24-hour reoxygenation. TUNEL staining and immunostaining for Müller and photoreceptor markers were used to detect which retinal cell types were damaged.. Culturing dissociated monkey retina cells for two weeks resulted in proliferation of Müller cells and maintenance of some rod and cone photoreceptor cells, as identified by vimentin, recoverin, and rhodopsin immunocytochemical staining. Hypoxia/reoxygenation increased the number of cells staining positive for TUNEL. Immunoblotting showed that the calpain-specific 145 kDa α-spectrin breakdown product (SBDP) increased in hypoxic cells, but no caspase-specific 120 kDa α-spectrin breakdown product was detected. TUNEL staining and proteolysis were significantly reduced in the retinal cells treated with 10 and 100 μM calpain inhibitor SNJ-1945. Caspase inhibitor, z-VAD, did not inhibit cell damage from hypoxia/reoxygenation. Intact pro-caspase-3 was in fact cleaved by activated calpain during hypoxia/reoxygenation to pre 29 kDa caspase-3 and 24 kDa inactive fragments. No 17 and 12 kDa fragments, which form the active caspase-3 hetero-dimer, were detected. Calpain-induced cleavage of caspase was inhibited by SNJ-1945.. Calpain, not caspase-3, was involved in hypoxic damage in cultured monkey retinal cells.

Topics: Animals; Apoptosis; Biomarkers; Calpain; Carbamates; Caspase 3; Caspase Inhibitors; Cell Count; Cell Proliferation; Cells, Cultured; Fluorescent Antibody Technique, Indirect; Hypoxia; Immunoblotting; In Situ Nick-End Labeling; Macaca mulatta; Neuroglia; Oligopeptides; Photoreceptor Cells, Vertebrate; Recoverin; Reperfusion Injury; Rhodopsin; Spectrin; Vimentin

Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels.

Topics: Analysis of Variance; Animals; Apoptosis; Calpain; Caspase 3; Cells, Cultured; Embryo, Mammalian; Excitatory Amino Acid Agonists; Glucose; Glutamic Acid; Hippocampus; Hypoxia; Infarction, Middle Cerebral Artery; Neurons; Rats; Rats, Wistar; Synaptic Vesicles; Transfection; Vesicular Glutamate Transport Protein 1; Vesicular Glutamate Transport Protein 2

Cancer cells which can survive and or proliferate in hypoxia may be resistant to anti-cancer treatment. In our previous work, we showed that we could group cell lines treated with severe hypoxia into either hypoxia-induced cell cycle arrest-sensitive or resistant phenotypes, and hypoxia-induced cell death (HCD)-sensitive or resistant phenotypes. We showed that the resistant phenotypes were associated with high levels of active-AKT in late hypoxia and sensitive cells were associated with decreased or undetectable levels of AKT in late hypoxia. We have now extended our findings to numerous other cell lines. We show that HCD and loss of AKT is cell density dependent, and both AKT1 and AKT2 isoforms are lost in late hypoxia. Loss of AKT is most likely due to regulated degradation, as transcription of AKT isoforms is unchanged in hypoxia, and AKT is not significantly translocated to the nucleus to account for its disappearance from cytoplasmic lysates. Interestingly, inhibitors of proteosome, calpain or caspase-mediated proteolysis did not significantly block AKT loss. Inhibition of autophagy using diverse lysosome-targeted autophagy inhibitors also did not block AKT loss, however autophagy inhibitors which block general PI3K activity, such as 3-methyladenine or LY294002, were effective inhibitors of AKT loss in late hypoxia. Interestingly, those inhibitors also blocked HCD in an HCD-sensitive cancer cell line. Inhibitors of proteolytic pathways which did not block AKT loss also did not block HCD in HeLa. Our investigations support a model by which AKT is a major switch involved in regulating hypoxia-induced cell death.

Topics: Adenine; Autophagy; Calpain; Chromones; Enzyme Inhibitors; Epithelial Cells; HeLa Cells; Humans; Hypoxia; Intracellular Signaling Peptides and Proteins; Morpholines; Oxygen; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Isoforms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

To explore the changes and regulation mechanism of dystropin and desmin under muscle injury without mechanic stress, 40 male Sprague-Dawley rats were randomly divided into 5 groups, which included normoxia control and hypoxia groups for 1, 2, 4 and 7 d with 10% O2. Two rats from each group were examined for sarcolemma integrity using Evans blue dye (EBD) and EBD-positive fiber typing by metachromatic dye-ATPase method. The rest six rats from each group were analyzed for the changes of protein content and gene expression using Western blot, RT-PCR and fluorescence assays. The results showed that the EBD-positive muscle fibers, mainly type IIA and type IIB, appeared at 1 d after hypoxia exposure. Both the ratio of EBD-positive cell and the mean fluorescence density were significantly higher in hypoxia groups than those in control group (P<0.05). The contents of dystrophin and desmin fluctuated after hypoxia exposure, increased at 1 d, decreased at 2 d, increased dramatically again at 4 d, and returned to a normal level at 7 d. Consistently, the gene expression began to increase significantly after 2 d. The total activity of calpain was significantly higher in hypoxia groups at 1, 4 and 7 d. Significantly higher levels of HSP70 and HSP90 were also observed at 4 and 7 d, respectively (P<0.05). These results suggest that the mechanical stress is not the only cause of damage of sarcolemma membrane integrity. In contrast to eccentric contraction, hypoxia-induced muscle damage is not accompanied by the loss of dystrophin and desmin. The types of muscle fibers recruited by motor units and the activities of calpain may be important in hypoxia-induced damage of sarcolemma membrane integrity.

Topics: Animals; Calpain; Desmin; Dystrophin; Hypoxia; Male; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Sarcolemma

Many factors contribute to nervous system dysfunction and failure to regenerate after injury or disease. Here, we describe a previously unrecognized mechanism for nervous system injury. We show that neuronal injury causes rapid, irreversible, and preferential proteolysis of the axon initial segment (AIS) cytoskeleton independently of cell death or axon degeneration, leading to loss of both ion channel clusters and neuronal polarity. Furthermore, we show this is caused by proteolysis of the AIS cytoskeletal proteins ankyrinG and betaIV spectrin by the calcium-dependent cysteine protease calpain. Importantly, calpain inhibition is sufficient to preserve the molecular organization of the AIS both in vitro and in vivo. We conclude that loss of AIS ion channel clusters and neuronal polarity are important contributors to neuronal dysfunction after injury, and that strategies to facilitate recovery must preserve or repair the AIS cytoskeleton.

Topics: Analysis of Variance; Animals; Axons; Calcium-Binding Proteins; Calpain; Cell Adhesion Molecules; Cell Death; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytoskeleton; Disease Models, Animal; Embryo, Mammalian; Glucose; Green Fluorescent Proteins; Hypoxia; Infarction, Middle Cerebral Artery; Mice; Mice, Inbred C57BL; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Transfection

Calpains are Ca(2+)-activated enzymes which cleave cytoskeletal and other proteins, contributing to neuronal damage in conditions of pathological intracellular Ca(2+) elevation, including stroke. However, the consequences of calpain overactivation have typically been observed hours after insult. To identify the earliest events attributable to calpain activation, and thus potentially isolate calpain substrates involved in acute neuronal damage, we dynamically recorded the effects of calpain inhibition in an in vitro model of stroke. Extracellular DC potentials and fEPSPs were monitored together with changes of light transmittance (as a measure of cell and mitochondrial swelling) and Rh 123 fluorescence (to monitor mitochondrial membrane potential; DeltaPsi(m)) in hippocampal slices obtained from P12-P17 rats. No differences were observed in the latencies of fEPSP disruption or onset of extracellular DC shifts associated with hypoxic spreading depression (HSD) evoked by oxygen-glucose deprivation (OGD) under control conditions or in the presence of calpain inhibitor III (MDL 28170). However, a significant difference was observed in transmitted light signals during OGD with calpain inhibition. Given the potential contribution of mitochondrial swelling to changes in light transmittance, these experiments were also conducted in the presence of cyclosporin A to block opening of the mitochondrial permeability transition pore (MPTP). Our results indicate that differences in OGD-induced changes of light transmittance in the presence of MDL 28170 are not likely the result of MPTP blockade or changes in dendritic beading. We propose that calpain inhibition may alter changes in light transmittance by limiting conformational changes of mitochondria.

Topics: Animals; Animals, Newborn; Brain Injuries; Calpain; Cortical Spreading Depression; Cysteine Proteinase Inhibitors; Dipeptides; Edema; Electric Stimulation; Excitatory Postsynaptic Potentials; Glucose; Hippocampus; Hypoxia; Membrane Potential, Mitochondrial; Rats; Rats, Wistar; Tissue Culture Techniques

Axonal injury in white matter is an important consequence of many acute neurological diseases including ischemia. A role for glutamate-mediated excitotoxicity is suggested by observations from in vitro and in situ models that AMPA/kainate blockers can reduce axonal injury. We assessed axonal vulnerability in primary murine neuronal cultures, with axons isolated from their cell bodies using a compartmented chamber design. Transient removal of oxygen and glucose in the axon compartment resulted in irreversible loss of axon length and neurofilament labeling. This injury was not prevented by addition of ionotropic glutamate receptor blockers and could not be reproduced by glutamate receptor agonists. However, hypoxic injury was prevented by blockade of voltage-gated sodium channels, inhibition of calpain and removal of extracellular calcium. These results suggest that isolated, unmyelinated axons are vulnerable to hypoxic injury which is mediated by influx of sodium and calcium but is independent of glutamate receptor activation.

Topics: Animals; Axons; Calcium; Calcium Signaling; Calpain; Cell Culture Techniques; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia; Hypoxia-Ischemia, Brain; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurotoxins; Receptors, Glutamate; Sodium; Sodium Channel Blockers; Sodium Channels; Wallerian Degeneration

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of micro-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially micro-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Calpain; Caspase Inhibitors; Caspases; Cell Death; Cell Line, Tumor; Cell Shape; Cisplatin; Cysteine Proteinase Inhibitors; Humans; Hypoxia; Poly(ADP-ribose) Polymerases; Tumor Suppressor Protein p53

Loss of mitochondrial membrane integrity and release of apoptogenic factors are a key step in the signaling cascade leading to neuronal cell death in various neurological disorders, including ischemic injury. Emerging evidence has suggested that the intramitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and promotes caspase-independent cell death induced by glutamate toxicity, oxidative stress, hypoxia, or ischemia. However, the mechanism by which AIF is released from mitochondria after neuronal injury is not fully understood. In this study, we identified calpain I as a direct activator of AIF release in neuronal cultures challenged with oxygen-glucose deprivation and in the rat model of transient global ischemia. Normally residing in both neuronal cytosol and mitochondrial intermembrane space, calpain I was found to be activated in neurons after ischemia and to cleave intramitochondrial AIF near its N terminus. The truncation of AIF by calpain activity appeared to be essential for its translocation from mitochondria to the nucleus, because neuronal transfection of the mutant AIF resistant to calpain cleavage was not released after oxygen-glucose deprivation. Adeno-associated virus-mediated overexpression of calpastatin, a specific calpain-inhibitory protein, or small interfering RNA-mediated knockdown of calpain I expression in neurons prevented ischemia-induced AIF translocation. Moreover, overexpression of calpastatin or knockdown of AIF expression conferred neuroprotection against cell death in neuronal cultures and in hippocampal CA1 neurons after transient global ischemia. Together, these results define calpain I-dependent AIF release as a novel signaling pathway that mediates neuronal cell death after cerebral ischemia.

Topics: Animals; Animals, Newborn; Apoptosis Inducing Factor; Brain; Calpain; Cells, Cultured; Disease Models, Animal; Electrophoresis, Gel, Pulsed-Field; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Glucose; Humans; Hypoxia; In Situ Nick-End Labeling; Mitochondria; Mitochondrial Proteins; Neurons; Rats; Rats, Sprague-Dawley; Time Factors; Transfection

In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC12 cells.

Topics: Animals; Blotting, Western; Calpain; Cell Differentiation; CREB-Binding Protein; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glucose; Hypoxia; L-Lactate Dehydrogenase; Nerve Growth Factor; Neural Inhibition; Neurons; Oncogene Protein v-akt; Oxygen; PC12 Cells; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rats; Serine; Spectrin; tau Proteins; Threonine; Time Factors

Alveolar hypoxia, a common feature of many respiratory disorders, has been previously reported to induce functional changes, particularly a decrease of transepithelial Na and fluid transport. In polarized epithelia, cytoskeleton plays a regulatory role in transcellular and paracellular transport of ions and fluid. We hypothesized that exposure to hypoxia could damage cytoskeleton organization, which in turn, may adversely affect ion and fluid transport. Primary rat alveolar epithelial cells (AEC) were exposed to either mild (3% O(2)) or severe (0.5% O(2)) hypoxia for 18 h or to normoxia (21% O(2)). First, mild and severe hypoxia induced a disorganization of actin, a major protein of the cytoskeleton, reflected by disruption of F-actin filaments. Second, alpha-spectrin, an apical cytoskeleton protein, which binds to actin cytoskeleton and Na transport proteins, was cleaved by hypoxia. Pretreatment of AEC by a caspase inhibitor (z-VAD-fmk; 90 microM) blunted hypoxia-induced spectrin cleavage as well as hypoxia-induced decrease in surface membrane alpha-ENaC and concomitantly induced a partial recovery of hypoxia-induced decrease of amiloride-sensitive Na transport at 3% O(2). Finally, tight junctions (TJs) proteins, which are linked to actin and are a determinant of paracellular permeability, were altered by mild and severe hypoxia: hypoxia induced a mislocalization of occludin from the TJ to cytoplasm and a decrease in zonula occludens-1 protein level. These modifications were associated with modest changes in paracellular permeability at 0.5% O(2,) as assessed by small 4-kD dextran flux and transepithelial resistance measurements. Together, these findings indicate that hypoxia disrupted cytoskeleton and TJ organization in AEC and may participate, at least in part, to hypoxia-induced decrease in Na transport.

Topics: Actins; Amino Acid Chloromethyl Ketones; Animals; Calpain; Cell Membrane; Cell Polarity; Cells, Cultured; Cytoskeleton; Epithelial Cells; Epithelial Sodium Channels; Hypoxia; Male; Membrane Proteins; Occludin; Patch-Clamp Techniques; Permeability; Phosphoproteins; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Spectrin; Tight Junctions; Zonula Occludens-1 Protein

One of the leading causes of blindness is retinal damage caused by the high intraocular pressure (IOP) in glaucoma. Previous studies in rats have suggested that the proteolytic enzyme calpain (EC 3.4.22.17) is involved in retinal cell death during ischemia and in acute high IOP. Ubiquitous, calcium-activated calpain-1 and -2 from monkey retina are highly homologous to rat calpains, although expression patterns in variants of tissue-specific calpain-3 are different between monkey and rodent retinas. Thus, the purpose of the present study was to investigate the involvement of calpain-induced proteolysis in retinal cell death in primates.. Calpain involvement in a simulated pathologic condition was examined by incubating monkey retinas in hypoxic conditions (95% N2 and 5% CO2) in RPMI medium without glucose. Endogenous tissue calpains were also directly activated in monkey and human retinal soluble proteins by incubating with 2.5 mM calcium. The resultant proteolysis of monkey retinal proteins was assessed by 2D electrophoresis (2-DE).. In hypoxic retina, leakage of lactate dehydrogenase (LDH) from retinas into the medium increased, indicating cell death. LDH leakage was partially inhibited by the calpain inhibitor SJA6017. Calpain autolysis was observed, and the calpain-preferred substrate alpha-spectrin was proteolyzed. In retinal soluble proteins incubated with calcium, a total of 15 spots from 2-DE of retinal soluble proteins were identified by mass spectrometry. Proteolysis of major proteins, vimentin, beta-tubulin, alpha-enolase, and Hsp70 were confirmed by immunoblot analysis. Activation of calpains and proteolysis of these substrates were inhibited by the calpain-specific inhibitor SJA6017.. Taken together, these results suggested that calpain activation in primate retinas could play an important role in cell death during hypoxia caused by elevated IOP from glaucoma.

Topics: Adult; Aged; Aged, 80 and over; Animals; Calcium; Calpain; Cell Death; Electrophoresis, Gel, Two-Dimensional; Humans; Hypoxia; Immunoblotting; Macaca mulatta; Middle Aged; Organ Culture Techniques; Retina

Our previous studies in retina on the mechanism for hypoxia-induced cell death suggested activation of a class of calcium-activated proteases known as calpains. This conclusion was based on data showing proteolysis of a calpain substrate alpha-spectrin, autolysis of activated calpain, and reduction of cell damage by calpain inhibitor SJA6017. Less is known about changes in downstream pathways after calpain activation. Thus, the purpose of the present investigation was to measure proteolysis of neuronal cytoskeletal proteins and apoptotic cell signaling factors during hypoxia-induced retinal cell death. Rat retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. Hypoxia was induced with 95% N2/5% CO2 without glucose. Immunoblotting was used to detect activation of calpain and proteolysis of substrates. Amounts of mRNA for calpain 1 and 2 were determined by quantitative PCR. Twelve times more calpain 2 mRNA than calpain 1 was present in retinas. Activation of calpain 2 and production of a calpain-specific alpha-spectrin breakdown product at 150 kDa were confirmed in hypoxic retinas. Further, pro-caspase-3 at 32 kDa was proteolyzed to a fragment at 30 kDa, tau protein was lost, and p35 was proteolyzed to p25 suggesting prolonged activation of cdk5. SJA6017 partially inhibited the production of these fragments. During hypoxia in rat retinas, calpains may be major proteases causing breakdown of neuronal proteins involved in apoptotic cell death. Calpain inhibitor SJA6017 may have potential for testing as a therapeutic agent against retinal pathologies such those caused by glaucoma, although future studies such as testing in in vivo animal models are required.

Topics: Animals; Calpain; Caspase 3; Caspases; Cell Death; Culture Media; Cytoskeleton; Hypoxia; Neurons; Oxygen; Phosphorylation; Rats; Retina; Spectrin; tau Proteins

Immature renal tubules are more tolerant to ischemia than mature renal tubules. Here we compared the developmental pattern for some cellular responses evoked by hypoxia and reoxygenation in renal proximal tubules from 10- and 40-day-old rats. Redistribution of Na(+)-K(+)-ATPase from the plasma membrane was studied by confocal microscopy techniques in primary cultured renal proximal tubular cells. The developmental expression of Na(+)-K(+)-ATPase, micro-calpain and heme oxygenase-1 was measured by RT-PCR techniques in rat renal cortex. In response to hypoxia Na(+)-K(+)-ATPase redistribution from the plasma membrane was almost 2-fold increased in cells isolated from mature kidneys compared with cells isolated from immature kidneys. Reoxygenation resulted in a complete reestablishment of Na(+)-K(+)-ATPase in the plasma membrane in the immature but not in the mature cells. The dissociation of Na(+)-K(+)-ATPase from the plasma membrane was associated with a reduced activity and a reduced expression of Na(+)-K(+)-ATPase in the mature but not in the immature tubular cells. The expression of micro-calpain, a factor shown to induce ischemic injury to proximal tubular cells, was significantly lower in the immature compared with the mature kidney, whereas the expression of heme oxygenase-1, a factor shown to protect from renal ischemic injury, was significantly higher in the immature kidney. The results help to explain the increased tolerance of the immature kidney to injury caused by ischemia and reperfusion.

Topics: Animals; Animals, Newborn; Base Sequence; Calpain; DNA Primers; Heme Oxygenase (Decyclizing); Hypoxia; Immunohistochemistry; Kidney Tubules, Proximal; Male; Microscopy, Confocal; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Sodium-Potassium-Exchanging ATPase

The goal of the present study was to determine the role of calpain in changes in plasma membrane permeability and cytoskeleton-associated paxillin, vinculin, talin, and alpha-actinin levels during acute renal cell death. The mitochondrial inhibitor antimycin A or hypoxia produced graded plasma membrane permeability in renal proximal tubules (RPTs), first allowing propidium iodide (PI, molecular mass 668 Da) influx and then lactate dehydrogenase (LDH, molecular mass 130 kDa) release. Cytoskeleton-associated paxillin levels decreased concomitantly with PI influx and before LDH release, whereas cytoskeleton-associated talin and vinculin levels decreased concomitantly with LDH release. Cytoskeleton-associated alpha-actinin levels did not change during antimycin A exposure or hypoxia. Purified micro-calpain cleaved paxillin, talin, vinculin, but not alpha-actinin. The dissimilar calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) or chloroacetic acid N'-[6,7-dichloro-4-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl] hydrazide (SJA7029) preserved cytoskeleton-associated paxillin, talin, and vinculin levels and prevented PI influx and LDH release in antimycin A-exposed or hypoxic RPTs. These results suggest that calpain mediates increased plasma membrane permeability and hydrolysis of cytoskeleton-associated paxillin, vinculin, and talin during renal cell death.

Topics: Acrylates; Actinin; Animals; Anti-Bacterial Agents; Antimycin A; Calpain; Cell Death; Cell Membrane Permeability; Coloring Agents; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dogs; Female; Hypoxia; Immunoblotting; Kidney; Paxillin; Phosphoproteins; Propidium; Rabbits; Swine; Talin; Vinculin

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.

Topics: Animals; Calpain; Eye Proteins; Hypoxia; Immunoblotting; In Vitro Techniques; Isoenzymes; L-Lactate Dehydrogenase; Rats; Rats, Sprague-Dawley; Retina; Spectrin; Time Factors

Calpains are cytosolic, Ca(2+)-activated, neutral cysteine proteases. Rabbit renal proximal tubule (RPT) cells express both mu- and m-calpain. Although multiple calpain inhibitors protect against RPT cell death, most calpain inhibitors lack specificity, membrane permeability, and/or potency. A group of novel catalytic site-directed calpain inhibitors, including chloroacetic acid N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl]hydrazide (SJA7019) and chloroacetic acid N'-(6,7-dichloro-4-phenyl-3-oxo-3,4-dihydroquinoxalin-2-yl) hydrazide (SJA7029), were identified to be potent calpain inhibitors in vitro. The goals of this study were to determine the action of these two compounds on 1) RPT calpain activity using fluorescein isothiocyanate-casein zymography, 2) antimycin A-induced RPT extracellular (45)Ca(2+) influx and cell death, and 3) hypoxia/reoxygenation-induced RPT cellular dysfunction and death. The results showed that the SJA compounds inhibited RPT mu- and m-calpain with equal potency (approximate IC(50), 30 microM) and efficacy, and blocked antimycin A-induced extracellular Ca(2+) influx and cell death. In addition, SJA7029 blocked cell death and allowed the recovery of mitochondrial function and active Na(+) transport in RPTs subjected to hypoxia/reoxygenation. In summary, the SJA compounds 1) were more potent inhibitors of calpains than catalytic site-directed peptide inhibitors in this model, 2) prevented extracellular Ca(2+) influx during the late phase of cell death, and 3) are true cytoprotectants and allow recovery of RPT cellular functions after injury.

Topics: Animals; Antimycin A; Calcium; Calpain; Cell Death; Cell Survival; Cysteine Proteinase Inhibitors; Extracellular Space; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hypoxia; Isoenzymes; Kidney; Kidney Tubules, Proximal; Oxygen Consumption; Rabbits

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Topics: Analysis of Variance; Animals; Blotting, Western; Calpain; Cell Death; Cells, Cultured; Cerebellum; Cerebral Cortex; Dipeptides; Dose-Response Relationship, Drug; Glucose; Humans; Hypoxia; Marine Toxins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neurotoxins; Oxocins; Rats; Rats, Sprague-Dawley; Spectrin; Time Factors

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."

Topics: Animals; Animals, Newborn; Apoptosis; Brain; Calpain; Carrier Proteins; Caspase 3; Caspases; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Enzyme Precursors; Female; Humans; Hypoxia; Immunoblotting; Immunohistochemistry; Inhibitory Concentration 50; Ischemia; Male; Microfilament Proteins; Protease Inhibitors; Rats; Rats, Wistar; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Substrate Specificity; Time Factors

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.

Topics: Amino Acid Transport Systems, Basic; Animals; Ankyrins; Arginine; Calpain; Carrier Proteins; Cell Membrane; Culture Techniques; Cysteine Proteinase Inhibitors; Endothelium, Vascular; Glycoproteins; Hypoxia; Immunoblotting; Membrane Proteins; Microfilament Proteins; Precipitin Tests; Pulmonary Artery; Reference Values; Swine

Calpains are ubiquitous Ca(2+)-activated neutral proteases that have been implicated in ischemic and traumatic CNS injury. Ischemia and trauma of central white matter are dependent on Ca2+ accumulation, and calpain overactivation likely plays a significant role in the pathogenesis. Adult rat optic nerves, representative central white matter tracts, were studied in an in vitro anoxic model. Functional recovery following 60 min of anoxia and reoxygenation was measured electrophysiologically. Calpain activation was assessed using western blots with antibodies against calpain-cleaved spectrin breakdown products. Sixty minutes of in vitro anoxia increased the amount of spectrin breakdown approximately 20-fold over control, with a further increase after reoxygenation to >70 times control, almost as much as 2 h of continuous anoxia. Blocking voltage-gated Na+ channels with tetrodotoxin or removing bath Ca2+ was highly neuroprotective electrophysiologically and resulted in a marked reduction of spectrin degradation. The membrane-permeable calpain inhibitors MDL 28,170 and calpain inhibitor-I (10-100 microM) were effective at reducing spectrin breakdown in anoxic and reoxygenated optic nerves, but no electrophysiological improvement was observed. We conclude that calpain activation is an important step in anoxic white matter injury, but inhibition of this Ca(2+)-dependent process in isolation does not improve functional outcome, probably because other deleterious Ca(2+)-activated pathways proceed unchecked.

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Electrophysiology; Glycoproteins; Hypoxia; Ion Channel Gating; Male; Optic Nerve; Oxygen; Rats; Rats, Long-Evans; Sodium Channel Blockers; Sodium Channels; Spectrin; Tetrodotoxin; Time Factors

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Hypoxia; In Vitro Techniques; Ionomycin; Ionophores; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Proteins; Rats

Topics: Animals; Calpain; Cell Membrane; Coumarins; Hypoxia; In Vitro Techniques; Ionomycin; Ionophores; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Oligopeptides; Rats; Substrate Specificity

Topics: Animals; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cells, Cultured; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; Fluorescent Dyes; Fura-2; Glutathione; Hypoxia; In Situ Hybridization, Fluorescence; Liver; Oligopeptides; Rats; 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

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.

Topics: Animals; Animals, Newborn; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Cell Membrane; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Female; Functional Laterality; Hypoxia; Male; Rats; Rats, Wistar; Up-Regulation

It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.

Topics: Animals; Calpain; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Glucose; Hippocampus; Hypoxia; Immunohistochemistry; In Vitro Techniques; Propidium; Rats; Rats, Wistar; Spectrin; Staining and Labeling

The role of the caspases, a newly discovered group of cysteine proteases, was investigated in a model of hypoxia-induced necrotic injury of rat renal proximal tubules. An assay for caspases in freshly isolated rat proximal tubules was developed. There was a 40% increase in tubular caspase activity after 15 min of hypoxia in association with increased cell membrane damage as indicated by a threefold increase in lactate dehydrogenase release. The specific caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB) attenuated the increase in caspase activity during 15 min of hypoxia and markedly decreased lactate dehydrogenase release in a dose-dependent manner. In the proximal tubules, Z-D-DCB also inhibited the hypoxia-induced increase in calpain activity, another cysteine protease. In contrast, when Z-D-DCB was added to purified calpain in vitro, there was no inhibition of calpain activity. The calpain inhibitor (2)-3-(4-iodophenyl)-2-mercapto-2-propenoic acid (PD150606) also inhibited the hypoxia-induced increase in caspase activity in proximal tubules, but did not inhibit the activity of purified caspase 1 in vitro. In these experiments, caspase activity was detected with the fluorescence substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC), which is preferentially cleaved by caspase 1. However, minimal caspase activity was detected with the fluorescence substrate Ac-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin (Ac-DEVD-AMC), which is cleaved by caspases 2, 3, and 7. The present study in proximal tubules demonstrates that (1) caspase inhibition protects against necrotic injury by inhibition of hypoxia-induced caspase activity; and (2) caspase 1 may be the caspase involved. Thus, although the role of caspases in apoptotic cell death is well established, this study provides new evidence that caspases contribute to necrotic cell death as well.

Topics: Animals; Aspartic Acid; Calpain; Caspase 1; Caspase Inhibitors; Caspases; Coumarins; Cysteine Proteinase Inhibitors; Hypoxia; Interleukin-1; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Male; Necrosis; Oligopeptides; Rats; Rats, Sprague-Dawley; Substrate Specificity

Stroke patients often experience a significant temporal delay between the onset of ischemia and the time to initiation of therapy. Thus, there is a need for neuroprotectants with a long therapeutic window of opportunity. The efficacy of a potent, central nervous system-penetrating calpain inhibitor (MDL 28,170) was evaluated in a temporary model of focal cerebral ischemia to determine the window of opportunity for intracellular protease inhibition.. An ex vivo brain protease inhibition assay established pharmacodynamic dosing parameters for MDL 28,170. Middle cerebral artery (MCA) occlusion was accomplished by advancing a monofilament through the internal carotid artery to the origin of the MCA. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium-stained brain sections.. Maximal inhibition of brain protease activity was observed 30 minutes after injection of MDL 28,170 with an estimated pharmacodynamic half-life of 2 hours. MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after MCA occlusion. A window of opportunity study was conducted to determine the maximal delay between the onset of ischemia and the initiation of efficacious therapy. MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay.. These data indicate that the therapeutic window of opportunity for calpain inhibition is at least 6 hours in a reversible focal cerebral ischemia model. This protection is observed despite the lethal hypoxic and excitotoxic challenge, suggesting that calpain activation may be an obligatory, downstream event in the ischemic cell death cascade.

Topics: Animals; Brain; Calpain; Carotid Artery, Internal; Cell Death; Cerebral Arterial Diseases; Cerebral Infarction; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Half-Life; Hypoxia; Image Processing, Computer-Assisted; Ischemic Attack, Transient; Male; Neuroprotective Agents; Neurotoxins; Rats; Rats, Wistar; Tetrazolium Salts; Time Factors

The effect of a novel neuroprotective compound, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride], on ischemia-induced fodrin breakdown was examined both in vitro and in vivo. The fodrin breakdown was measured by western blot followed by a densitometric analysis. In slices of the rat cerebral cortex, a pronounced fodrin breakdown was observed under hypoxic and hypoglycemic conditions. The enhancement of fodrin breakdown was completely blocked by omission of extracellular Ca2+ and significantly inhibited by calpain inhibitors such as E-64 and calpain inhibitor-I, thereby suggesting that the fodrin breakdown induced by hypoxia/hypoglycemia is due to the activation of Ca2+-stimulated neutral protease calpain. NS-7 (1-30 microM) produced a concentration-dependent inhibition of hypoxia/hypoglycemia-induced fodrin breakdown. In rats with unilateral middle cerebral artery occlusion (MCAO), a pronounced fodrin breakdown was observed in the cerebral cortex and striatum, although the time course for the development of the fodrin breakdown was much slower in the cerebral cortex than in the striatum. NS-7 (0.5 mg/kg i.v.), when injected immediately after MCAO, suppressed not only the fodrin breakdown but also the infarction in the cerebral cortex. From these results it is suggested that inhibition of calpain activation is implicated in the neuroprotective action of NS-7.

Topics: Animals; Arterial Occlusive Diseases; Brain Ischemia; Calcium; Calpain; Carrier Proteins; Cerebral Cortex; Cerebral Infarction; Enzyme Activation; Glucose; Hypoglycemia; Hypoxia; Male; Microfilament Proteins; Neostriatum; Nerve Tissue Proteins; Neuroprotective Agents; Organ Culture Techniques; Oxygen; Piperazines; Pyrimidines; Rats; Rats, Sprague-Dawley

The role of calcium-activated proteolysis in hypoxic neuronal injury was investigated using an in vitro slice model of moderate hypoxia that mimics many features of an ischemic penumbra. The calpain inhibitor, MDL28170, significantly improved the recovery of synaptic responses in hippocampal slices following prolonged, moderate hypoxia without hypoxic depolarization. This finding further implicates calpain-mediated proteolysis in the development of neuronal injury following moderate metabolic challenge such as occurs in regions of partial ischemia.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Excitatory Postsynaptic Potentials; Hippocampus; Hypoxia; Male; Neurons; Rats; Rats, Sprague-Dawley; Synapses

Calpain proteases contribute to hepatocyte necrosis during anoxia. Our aim was to ascertain the mechanism causing calpain activation during anoxia. In rat hepatocytes, a twofold increase in calpain activity occurred despite the lack of an increase in cytosolic Ca2+ concentration ([Ca2+]i). The increase in calpain activity was not associated with an increase in calpain mRNA or a decrease in calpastatin mRNA expression. Because phospholipid degradation products generated by phospholipases can activate calpains at physiological [Ca2+]i, we determined the effect of phospholipase inhibitors and activators on calpain activity. Pretreatment of hepatocytes with fluphenazine, a phospholipase inhibitor, decreased calpain activation and improved cell survival. Melittin, a phospholipase A2 activator, increased calpain activity and potentiated cell killing. Finally, phospholipid degradation preceded the increase in calpain activity. Thus the enhanced calpain activity occurring in hepatocytes during anoxia 1) is regulated at the posttranslational level and 2) appears to be dependent on phospholipase activity. These data suggest a novel cascade for degradative hydrolase activity during hepatocyte necrosis by anoxia with phospholipase-mediated activation of calpains.

Topics: Animals; Calcium; Calpain; Cells, Cultured; Hydrolases; Hypoxia; In Situ Hybridization, Fluorescence; Intracellular Membranes; Liver; Necrosis; Phospholipases; Phospholipids; Protein Biosynthesis; Rats; Transcription, Genetic

The effect of the newly developed, nonpeptide, calpain inhibitor, PD 150606, on hypoxia and ionomycin-induced increases in calpain activity in rat proximal tubules (PT) was determined. PD150606 inhibited both hypoxia and ionomycin-induced calpain activity as determined by the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC). This decrease in calpain activity was accompanied by dose-dependent cytoprotection against hypoxia and ionomycin-induced cell membrane damage. PD150606 had no effect on cathepsin B and L activity in PT as measured by the fluorescent substrate, benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl coumarin (Z-Phe-Arg-AMC). The effects of low intracellular pH (pHi) or low free cytosolic calcium [Ca2+]i on this hypoxia-induced calpain activity were also determined. Both low pHi and low [Ca2+]i attenuated the hypoxia-induced increase in calpain activity. This attenuation of calpain activity was observed early before hypoxia-induced membrane damage and was associated with marked reduction in the typical pattern of hypoxia-induced cell membrane damage observed in this model. To identify the isoform of calpain activated in rat proximal tubules, normoxic, hypoxic and ionomycin treated tubules were fractionated by MONO-Q anion exchange chromatography and the fractions were assayed for calpain activity. A single peak of calpain activity characteristic of mu-calpain was found. The calcium dependency of the calpain activity was in the nanomolar range, further confirming that the activity was the low Ca(2+)-sensitive mu-calpain. The present study suggests that in rat proximal tubules: (1) PD 150606 is a specific inhibitor of calpain and not cathepsins B and L; (2) the cytoprotective effects of low pHi and low [Ca2+]i are mediated, at least in part, by inhibition of calpain activity; and (3) the predominant active form of calpain is the isoenzyme mu-calpain.

Topics: Acrylates; Analysis of Variance; Animals; Calcium; Calpain; Cathepsins; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Male; Rats; Rats, Sprague-Dawley

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min). Ipsi-and contralateral parietal cerebral cortex was assayed with Western blotting for fodrin breakdown product (FBDP). Calpain immunoreactivity was assayed in the cytosolic fraction (CF) and the membrane and microsomal fraction (MMF). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This relative redistribution of calpains coincided with the appearance of FBDP in the left, ipsilateral hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP could be detected in the right, contralateral hemisphere, indicating that although redistribution of calpains occurred, hypoxia per se did not suffice to initiate fodrin degradation in this model of neonatal hypoxic-ischemia.

Topics: Animals; Animals, Newborn; Antibodies; Blotting, Western; Brain Chemistry; Brain Ischemia; Calpain; Carrier Proteins; Disease Models, Animal; Female; Hypoxia; Male; Microfilament Proteins; Nerve Tissue Proteins; Rats; Rats, Wistar

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

Topics: Animals; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Dipeptides; Endopeptidases; Hypoxia; In Vitro Techniques; Ionomycin; Kidney Cortex; Kidney Tubules, Proximal; Kinetics; Male; Rats; Rats, Sprague-Dawley; Reference Values; Serine Proteinase Inhibitors

Rabbits were subjected to hypoxia (5% O2) for up to 90 min and allowed to recover for a maximum of 4 days. Hippocampus homogenate was assayed for fodrin breakdown product (BDP). After separation into a nuclear and mitochondrial fraction (NMF), a membrane and microsomal fraction (MMF), and a cytosolic fraction (CF), samples were assayed for mu-calpain, m-calpain, and calpastatin immunoreactivity. Calpain and calpastatin immunoreactivity decreased in the NMF and CF but increased in the MMF during hypoxia and short-term recovery. This translocation occurred in parallel with the increase in fodrin BDP. Because the increase in the MMF was not large enough to explain the decrease in the other two fractions, it was assumed that the translocation and activation was accompanied by a reduction in the total amounts of calpains and calpastatin. Glucocorticoid pretreatment (beta-methasone, 0.4 mg x kg-1 x day-1) for 7 days produced a decrease in the ratio of activated mu-calpain in all three fractions in nearly all samples before, during, and after hypoxia, compared with untreated animals. Glucocorticoid pretreatment also prevented the increase in fodrin BDP that occurred in untreated animals during hypoxia and short-term recovery, indicating impairment of calpain activation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Membrane; Cell Nucleus; Cytosol; Glucocorticoids; Hippocampus; Hypoxia; Microfilament Proteins; Microsomes; Mitochondria; Rabbits; Subcellular Fractions

The mechanism by which glycine protects against hepatocyte death during anoxia remains unclear. Nonlysosomal proteolysis, including calpain proteolysis, has been implicated as a mechanism of lethal cell injury. However, the effect of glycine on nonlysosomal proteolysis is unknown. The aim of this study was to ascertain if glycine cytoprotection is associated with inhibition of nonlysosomal proteolysis.. Rat hepatocyte suspensions were rendered anoxic using an anaerobic chamber. Cell viability was measured by propidium iodide fluorometry. Nonlysosomal protease activity was quantitated by the release of trichloroacetic acid-soluble free amines or tyrosine. Calpain protease activity was measured using a fluorogenic substrate.. Glycine and alanine (but not valine) markedly improved cell viability during anoxia in a concentration-dependent manner. During anoxia, the majority of nonlysosomal proteolysis (60%) was dependent on extracellular Ca2+. Glycine only inhibited that portion of nonlysosomal proteolysis that was dependent on extracellular Ca2+. Amino acids inhibited the anoxia-stimulated increase in calpain protease activity with the same specificity and concentration-dependence observed for cytoprotection. Glycine was more potent in directly inhibiting purified m-calpain as compared with mu-calpain protease activity.. Glycine may exert its cytoprotective activity during lethal anoxic hepatocyte injury, in part by inhibiting Ca(2+)-dependent degradative, nonlysosomal proteases, including calpains.

Topics: Animals; Calcium; Calpain; Cell Membrane Permeability; Cells, Cultured; Glycine; Hypoxia; Liver; Lysosomes; Male; Peptide Hydrolases; Protease Inhibitors; Rats; Rats, Sprague-Dawley

The contribution of metabolic energy to the degradation of intracellular proteins in skeletal muscle was investigated. Isolated chick skeletal muscles deprived of oxygen and muscles incubated in buffer under nonphysiological conditions containing inhibitors of glycolysis and mitochondrial respiration had lower concentrations or undetectable levels of ATP and faster rates of proteolysis. Both total protein breakdown and the breakdown of myofibrillar proteins were stimulated 35-124% in ATP-depleted tissues. However, ATP-depleted muscles incubated in buffer to which no Ca2+ was added showed slower rates of total protein breakdown and no significant change in myofibrillar proteolysis compared with control muscles. Trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), a compound that inhibits the calpains and the lysosomal cysteine proteases, completely blocked the Ca(2+)-stimulated breakdown of nonmyofibrillar and myofibrillar proteins in ATP-depleted muscles. However, Ca(2+)-stimulated proteolysis was not inhibited in ATP-depleted muscles incubated with weak bases to prevent lysosome function. These data suggest that intracellular proteins can be degraded in skeletal muscle in the absence of metabolic energy and that the calpains play a major role in the enhanced proteolysis in skeletal muscles depleted of ATP.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Calcimycin; Calcium; Calpain; Chickens; Cysteine Proteinase Inhibitors; Energy Metabolism; Hypoxia; Ionomycin; Male; Muscle Proteins; Peptide Hydrolases

In vitro hippocampal slices from adult rats were subjected to transient hypoxia in the presence of a cell-penetrating, calpain inhibitor (Cbz-Val-Phe-H; MDL-28170). The posthypoxic recovery of synaptic potentials was greatly improved in protease inhibitor-treated slices relative to control slices. These findings support a role for calcium-activated proteolysis in the process of hypoxic pathophysiology.

Topics: Animals; Calpain; Cell Death; Cell Membrane Permeability; Dipeptides; Hippocampus; Hypoxia; Rats; Rats, Inbred Strains

Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons.

Topics: Animals; Calpain; Cerebral Ventricles; Electrophysiology; Endopeptidases; Evoked Potentials; Gerbillinae; Hippocampus; Hypoxia; Infusions, Parenteral; Ischemic Attack, Transient; Leupeptins; Neurons; Spectrin; Synapses