calpain and aloxistatin

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. LIS1 (official symbol PAFAH1B1, for platelet-activating factor acetylhydrolase, isoform 1b, subunit 1) was identified as the gene mutated in individuals with lissencephaly, and it was found to regulate cytoplasmic dynein function and localization. Here we show that inhibition or knockdown of calpains protects LIS1 from proteolysis, resulting in the augmentation of LIS1 amounts in Lis1(+/-) mouse embryonic fibroblast cells and rescue of the aberrant distribution of cytoplasmic dynein, mitochondria and beta-COP-positive vesicles. We also show that calpain inhibitors improve neuronal migration of Lis1(+/-) cerebellar granular neurons. Intraperitoneal injection of the calpain inhibitor ALLN to pregnant Lis1(+/-) dams rescued apoptotic neuronal cell death and neuronal migration defects in Lis1(+/-) offspring. Furthermore, in utero knockdown of calpain by short hairpin RNA rescued defective cortical layering in Lis1(+/-) mice. Thus, calpain inhibition is a potential therapeutic intervention for lissencephaly.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Animals; Calpain; Cell Movement; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Disease Models, Animal; Dyneins; Embryo, Mammalian; Female; Fibroblasts; Gene Expression Regulation, Developmental; Leucine; Leupeptins; Lissencephaly; Mice; Mice, Knockout; Microtubule-Associated Proteins; Models, Neurological; Neurons; Phenotype; Pregnancy

Calpains are calcium-dependent enzymes that determine the fate of proteins through regulated proteolytic activity. Calpains have been linked to the modulation of memory and are key to the pathogenesis of Alzheimer disease (AD). When abnormally activated, calpains can also initiate degradation of proteins essential for neuronal survival. Here we show that calpain inhibition through E64, a cysteine protease inhibitor, and the highly specific calpain inhibitor BDA-410 restored normal synaptic function both in hippocampal cultures and in hippocampal slices from the APP/PS1 mouse, an animal model of AD. Calpain inhibition also improved spatial-working memory and associative fear memory in APP/PS1 mice. These beneficial effects of the calpain inhibitors were associated with restoration of normal phosphorylation levels of the transcription factor CREB and involved redistribution of the synaptic protein synapsin I. Thus, calpain inhibition may prove useful in the alleviation of memory loss in AD.

Topics: Alzheimer Disease; Animals; Calpain; Cells, Cultured; Disease Models, Animal; Glycoproteins; Hippocampus; Homozygote; Immunohistochemistry; Leucine; Memory; Mice; Mice, Transgenic; Models, Neurological; Synaptic Transmission

To demonstrate calpain involvement in neurodegeneration in rat spinal cord injury (SCI), we examined SCI segments for DNA fragmentation, neurons for calpain overexpression, neuronal death, and neuroprotection with calpain inhibitor (E-64-d). After the induction of SCI (40 g cm force) on T12, rats were treated within 15 min with vehicle (DMSO) or E-64-d. Sham animals underwent laminectomy only. Animals were sacrificed at 24 h, and five 1-cm long spinal cord segments were collected: two rostral (S1 and S2), one lesion (S3), and two caudal segments (S4 and S5). Agarose gel electrophoresis of DNA samples isolated from the SCI segments showed both random and internucleosomal DNA fragmentation indicating occurrence of necrosis as well as apoptosis mostly in the lesion, moderately in caudal, and slightly in rostral segments from SCI rats. Treatment of SCI rats with E-64-d (1 mg/kg) reduced DNA fragmentation in all segments. The lesion and adjacent caudal segments (S3 and S4) were further investigated by in situ double-immunofluorescent labelings that showed increase in calpain expression in neurons in SCI rats and decrease in calpain expression in SCI rats treated with E-64-d. In situ combined TUNEL and double-immunofluorescent labelings directly detected co-localization of neuronal death and calpain overexpressin in SCI rats treated with only vehicle while attenuation of neuronal death in SCI rats treated with E-64-d. Previous studies from our laboratory indirectly showed neuroprotective effect of E-64-d in SCI rats. Our current results provide direct in situ evidence for calpain involvement in neuronal death and neuroprotective efficacy of E-64-d in lesion and penumbra in SCI rats.

Topics: Animals; Apoptosis; Calpain; Cell Death; Cysteine Proteinase Inhibitors; DNA Fragmentation; Fluorescent Antibody Technique; In Situ Nick-End Labeling; Leucine; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries

Matrix metalloproteinases (MMPs) and cysteine proteases (calpain and cathepsin B) play an important role in cell death and are upregulated after focal cerebral ischemia. Because there is a significant interaction between MMP-9 with calpain and cathepsin B, we investigated the role of E64d (a calpain and cathepsin B inhibitor) on MMP-9 activation in the rat focal ischemia model.. Male Sprague-Dawley rats were subjected to 2 hours of middle cerebral artery occlusion by using the suture insertion method followed by 22 hours of reperfusion. In the treatment group, a single dose of E64d (5 mg/kg IP) was administrated 30 minutes before the induction of focal ischemia, whereas the nontreatment group received dimethyl sulfoxide only. The neurological deficits, infarct volumes, Evans blue extravasation, brain edema, and MMP-9 activation in the brain were determined.. Pretreatment with E64d produced a significant reduction in the cerebral infarction volume (353.1+/-19.8 versus 210.3+/-23.7 mm3) and the neurological deficits. Immunofluorescence studies showed MMP-9, calpain, and cathepsin B activation colocalized to both neurons and the neurovascular endothelial cells after ischemia, which was reduced by E64d.. These results suggest that E64d treatment provides a neuroprotective effect to rats after transient focal cerebral ischemia by inhibiting the upregulation of MMP-9.

Topics: Animals; Blood-Brain Barrier; Brain Edema; Calpain; Cathepsin B; Cerebral Hemorrhage; Cysteine Proteinase Inhibitors; Drug Evaluation, Preclinical; Enzyme Activation; Enzyme Induction; Evans Blue; Extravasation of Diagnostic and Therapeutic Materials; Infarction, Middle Cerebral Artery; Leucine; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Nerve Tissue Proteins; Neuroprotective Agents; Premedication; Rats; Rats, Sprague-Dawley; Reperfusion; Single-Blind Method

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a mu-calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood-brain barrier permeability, histology, and immunohistochemistry. The E64d-treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d-treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase-3 activation and apoptotic changes in both neurons and endothelial cells in E64d-treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.

Topics: Analysis of Variance; Animals; Apoptosis; Brain Edema; Brain Infarction; Brain Ischemia; Calpain; Caspase 3; Caspases; Cathepsins; Endothelium, Vascular; Functional Laterality; Gene Expression; Immunohistochemistry; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Leucine; Male; Neurologic Examination; Neurons; Neuroprotective Agents; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Tetrazolium Salts

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs.

Topics: Acrylates; Animals; Calpain; Cathepsins; CDC2 Protein Kinase; Cell Cycle; Cell Nucleus; Cyclin B; Cysteine Proteinase Inhibitors; Cytoplasm; DNA Replication; Embryo, Nonmammalian; Leucine; Mitosis; Sea Urchins; Tissue Distribution

Calpain, a calcium-dependent cysteine proteinase, has been reported to participate in the pathophysiology of rheumatoid arthritis (RA). The aim of this study is to investigate the therapeutic efficacy of calpain-inhibitory compounds in an animal model of RA and to clarify the underlying mechanisms in vivo and in vitro. Arthritis was induced in BALB/c mice with anti-type II collagen mAbs and LPS, and the mice were treated intra-peritoneally with a high dose (9 mg kg(-1) per day) or low dose (3 mg kg(-1) per day) of E-64-d (a membrane-permeable cysteine proteinase inhibitor) or control diluent. As a result, a high dose of E-64-d significantly alleviated the clinical arthritis and the histopathological findings, compared with the control diluent, although a low dose of E-64-d did not have a significant effect. Next, we evaluated the effects of E-64-d on cytokine mRNA expression at the inflamed joints by quantitative reverse transcription-PCR. High dose of E-64-d significantly decreased IL-6 and IL-1beta mRNA levels at the inflamed joints. The regulatory effects of E-64-d on cytokine production were also confirmed in vitro, using a synovial cell line (E11) and crude synoviocytes derived from RA patients. These results suggest the key roles of calpain in the pathophysiology of arthritis and that calpain-inhibitory compounds might be applicable to the treatment of arthritic diseases such as RA.

Topics: Animals; Arthritis, Experimental; Calpain; Cell Line; Cytokines; Female; Gene Expression; Leucine; Mice; Mice, Inbred BALB C; RNA, Messenger

The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia.. Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time.. In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01).. In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Humans; Hypertrophy, Left Ventricular; Leucine; Male; Nephrectomy; Oligopeptides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Uremia

Genetic variation in the gene for a cytosolic cysteine protease, calpain-10, increases the susceptibility to type 2 diabetes apparently by altering levels of gene expression. In view of the importance of altered beta-cell function in the pathophysiology of type 2 diabetes, the present study was undertaken to define the effects on insulin secretion of exposing pancreatic islets to calpain inhibitors for 48 hours. Exposure of mouse islets to calpain inhibitors (ALLN, ALLM, E-64-d, MDL 18270, and PD147631) of different structure and mechanism of action for 48 hours reversibly suppresses glucose-induced insulin secretion by 40% to 80%. Exposure of islets to inhibitors of other proteases, ie, cathepsin B and proteasome, did not affect insulin secretion. The 48-hour incubation with calpain inhibitors also attenuates insulin secretory responses to the mitochondrial fuel alpha-ketoisocaproate (KIC). The same incubation also suppresses glucose metabolism and intracellular calcium ([Ca(2+)](i)) responses to glucose or KIC in islets. In summary, long-term inhibition of islet calpain activity attenuates insulin secretion possibly by limiting the rate of glucose metabolism. A reduction of calpain activity in islet could contribute to the development of beta-cell failure in type 2 diabetes thereby providing a link between genetic susceptibility to diabetes and the pathophysiologic manifestations of the disease.

Topics: Animals; Calcium; Calpain; Cell Separation; Cysteine Proteinase Inhibitors; Dipeptides; Energy Metabolism; Glucose; In Vitro Techniques; Insulin; Islets of Langerhans; Leucine; Mice; Mice, Inbred C57BL; Mitochondria; NADP; Oxidation-Reduction

Spinal cord injury (SCI) is associated with progressive neurodegeneration and dysfunction. Multiple cellular and molecular mechanisms are involved in this pathogenesis. In particular, the activation of proteases following trauma can cause apoptosis in the spinal cord. Calpain, a calcium-dependent cysteine protease, plays a major role in apoptosis following trauma. We identified apoptosis and decrease in transcription of the genes for proteolipid protein (PLP) and myelin basic protein (MBP) in five 1-cm long spinal cord segments (S1, distant rostral; S2, near rostral; S3, lesion; S4, near caudal; and S5, distant caudal) 24 h after induction of SCI (40 g.cm force) in rats by weight-drop method. Sham rats underwent laminectomy and did not receive injury. Internucleosomal DNA fragmentation occurred prominently in the lesion (S3), moderately in near segments (S2 and S4), and slightly in distant segments (S1 and S5) of injured rats, indicating the occurrence of apoptosis in the lesion and penumbra. Levels of transcription of PLP and MBP were reduced highly in the lesion and moderately in near segments, suggesting that apoptotic loss of cells impaired biosynthesis of two important structural components of myelin. Immediate administration of the calpain inhibitor E-64-d (1 mg/kg) to injured rats prevented apoptosis and restored transcription of these genes, indicating the therapeutic efficacy of calpain inhibitor for treatment of SCI.

Topics: Animals; Apoptosis; Calpain; DNA Fragmentation; Electrophoresis, Agar Gel; Leucine; Myelin Basic Protein; Myelin Proteolipid Protein; Nucleosomes; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord Injuries; Transcription, Genetic

Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21cip1/waf1 (p21cip1) and p27kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21cip1, fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21cip1 levels after HCMV infection were investigated by measuring p21cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21cip1 RNA and protein levels suggested that the degradation of p21cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21cip1 in mock-infected cells, but MG132 was much less effective in protecting p21cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21cip1 in a concentration-dependent manner. To verify that p21cip1 was a substrate for calpain, purified recombinant p21cip1 was incubated with either m-calpain or mu-calpain, which resulted in rapid proteolysis of p21cip1. E64d inhibited the proteolysis of p21cip1 catalyzed by either m-calpain or mu-calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in

Topics: Amino Acid Motifs; Calcium-Binding Proteins; Calpain; Cell Cycle; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cytomegalovirus; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Leucine; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; RNA, Messenger; Time Factors; Ubiquitins

Degradation of cytoskeletal proteins by calpain, a Ca(2+)-dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g-cm force) SCI in rats was induced at T12 by a standardized weight-drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24-h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell-permeable calpain inhibitor E-64-d (1 mg/kg). Following 24-h treatment, a 5-cm-long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68-kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E-64-d-treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E-64-d providing neuroprotective benefit.

Topics: Animals; Apoptosis; Calpain; Cell Death; Cycloheximide; Female; Glycoproteins; Leucine; Protease Inhibitors; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries

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

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

We have reported previously that the abnormally down-regulated protein kinase C (PKC) causes cellular dysfunction observed in natural killer (NK) cells, polymorphonuclear leucocytes (PMNs) and fibroblasts from beige mouse, an animal model of Chediak-Higashi syndrome (CHS). Here we show that the abnormal down-regulation of PKC activity also occurs in Epstein-Barr (EB) virus-transformed cell lines from CHS patients. When CHS cell lines were stimulated with concanavalin A (Con A) for 20 min, the membrane-bound PKC activity declined markedly, whereas that in control cell lines increased. We found that E-64-d, which protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and corrected the increased Con A cap formation to almost normal levels in CHS cell lines. We confirmed that the dysregulation of PKC activity also occurred in peripheral blood mononuclear leucocytes (PBMC) from CHS patients and that E-64-d corrected both the declined PKC activity and increased Con A cap formation. E-64-d also corrected the reduced lysosomal elastase and cathepsin G activity in CHS cell lines. In contrast, chelerythrin, a specific inhibitor of PKC, and C2-ceramide, which promotes PKC breakdown induced by calpain, increased Con A cap formation and inhibited both elastase and cathepsin G activity in normal cell lines. Moreover, we found that ceramide production in CHS cell lines increased significantly after Con A stimulation, which coincides with our previous observation in fibroblasts from CHS mice. These results suggest an association between ceramide-induced PKC down-regulation and the cellular dysfunctions in CHS.

Topics: Calpain; Cathepsin G; Cathepsins; Cell Line, Transformed; Ceramides; Chediak-Higashi Syndrome; Child; Concanavalin A; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Antagonism; Female; Humans; Leucine; Leukocytes, Mononuclear; Lysosomes; Male; Pancreatic Elastase; Protein Kinase C; Receptor Aggregation; Serine Endopeptidases; Sphingomyelin Phosphodiesterase

In essential hypertension (EHT) the presence of a metabolic syndrome increases the risk of cardiovascular disease. A cell membrane abnormality is implicated but its role in cardiovascular disease is unclear. Neutrophil accumulation, which occurs by beta2-integrin (CD11b/CD18) expression, followed by release of proinflammatory factors from primary vesicles is an important factor in vascular damage. CD11b and CD69 expression on neutrophils from normal controls and EHT patients was determined by fluorescence-activated cell scanning. Neutrophils were activated with phorbol myristate acetate (PMA). Protein kinase C (PKC) and calpain were inhibited with bisindolylmaleimide and E64d, respectively. In EHT patients CD11b was not increased on neutrophils at rest. However, EHT neutrophils more readily expressed CD11b on incubation in phosphate-buffered saline and more cells went on to exocytose primary granules indicated by expression of CD69. Stimulation with PMA caused more rapid activation in EHT neutrophils with expression of CD11b, followed rapidly by exocytosis of primary granules. Bisindolylmaleimide slowed but did not prevent CD11b expression, which, together with primary granule exocytosis, continued to be faster in EHT neutrophils. E64d also slowed but did not prevent either CD11b expression or primary granule exocytosis, but this inhibitor did abolish the difference between NC and EHT neutrophils. The membrane abnormality in EHT may contribute to cardiovascular risk by increasing the rate of vesicle fusion with the cell membrane to increase neutrophil accumulation and release of inflammatory agents at sites of vascular damage. Calpain activation may be the rate-limiting component that is abnormal.

Topics: Adult; Albuminuria; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Blood Vessels; Calpain; Carcinogens; Cell Fusion; Cell Membrane; Cysteine Proteinase Inhibitors; Cytoplasmic Granules; Enzyme Inhibitors; Female; Humans; Hypertension; Indoles; Lectins, C-Type; Leucine; Lipids; Macrophage-1 Antigen; Male; Maleimides; Metabolic Syndrome; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Tetradecanoylphorbol Acetate

Upregulation of calpain, a Ca(2+)-activated cysteine protease, has been implicated in apoptosis and tissue degeneration in spinal cord injury (SCI) that over time spreads from the site of injury to the surrounding regions. We examined calpain content and activity, regulation of immediate early genes (IEGs) such as c-jun and c-fos, reactive astrogliosis as the expression of glial fibrillary acidic protein (GFAP), and apoptosis-related features such as caspase-3 mRNA expression and internucleosomal DNA fragmentation in 1-cm long spinal cord segments (S1, distant rostral; S2, adjacent rostral; S3, lesion or injury; S4, adjacent caudal; and S5, distant caudal) following SCI in rats. Calpain content and production of 150 kD calpain-cleaved alpha-fodrin fragment, expression of IEGs, reactive astrogliosis, and apoptotic features were highly increased in the lesion (S3), moderately in adjacent areas (S2 and S4), and slightly in distant areas (S1 and S5) in SCI rats when compared to sham animals. Administration of the calpain-specific inhibitor E-64-d (1 mg/kg) to SCI rats continuously for 24 h inhibited calpain activity and other factors contributing to apoptosis in the lesion and surrounding areas, indicating that calpain played a key role in the pathophysiology of SCI. The results obtained from this animal model of SCI suggest that calpain inhibitor can provide neuroprotection in patients with SCI.

Topics: Animals; Apoptosis; Astrocytes; Calpain; Carrier Proteins; Caspase 3; Caspases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Female; Genes, Immediate-Early; Glial Fibrillary Acidic Protein; Gliosis; Leucine; Microfilament Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Spinal Cord Injuries

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA Primers; Female; Gene Expression; Leucine; Neurofilament Proteins; Nucleosomes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord Injuries

Calpain content was investigated in the lesion of rat spinal cord at 1, 4, 24, and 72 h following injury induced by the weight-drop (40 g-cm force) technique. Calpain content was increased in the lesion, and was highest at 24 h following injury. microCalpain mRNA level in the lesion was increased by 58.4% (p = 0.0135) at 24 h following trauma, compared to sham. Alterations in mRNA expression in the lesion increased bax/bcl-2 ratio by 20.8% (p = 0.0395) at this time point, indicating a commitment to apoptosis. Therapeutic effect of the calpain inhibitor E-64-d (1 mg/kg) was studied in SCI rats following administration for 24 h. Internucleosomal DNA fragmentation (apoptosis) was observed in SCI rats, but not in sham or E-64-d treated rats. These results indicate a new information that E-64-d has the therapeutic potential for inhibiting apoptosis in SCI.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Cysteine Proteinase Inhibitors; DNA Fragmentation; Female; Gene Expression Regulation, Enzymologic; Leucine; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Spinal Cord Injuries; Transcription, Genetic

Ceramide has been recognized as an important second messenger in intracellular signaling. We demonstrate here that ceramide promotes the down-regulation of protein kinase C (PKC) activity in phorbol ester-stimulated murine polymorphonuclear leukocytes (PMNs). As reported previously, treatment of PMNs with phorbol ester caused a translocation of PKC from the cytosolic to the membrane fractions. When PMNs were pretreated with cell-permeable ceramide analogue, C2-ceramide, the membrane-associated PKC activity was rapidly down-regulated by phorbol ester stimulation. E64-d, a potent inhibitor of calpain which proteolyzes PKC, eliminated the rapid down-regulation of PKC activity. By hydroxyapatite column chromatography and Western blotting, the predominant PKC isoform was PKC beta with a small amount of PKC alpha in murine PMNs. We found that ceramide strikingly promoted calpain-mediated proteolysis of PKC beta in vitro. Ceramide was also shown to inhibit [3H]phorbol 12,13-dibutyrate(PDBu) binding to PKC beta. Moreover, we show that ceramide stimulates PKC beta autophosphorylation. These results suggest that ceramide directly activates PKC beta and promotes calpain-mediated proteolysis in murine PMNs.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Down-Regulation; Isoenzymes; Leucine; Mice; Mice, Inbred C57BL; Neutrophils; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Protein Kinase C beta; Sphingosine

To determine whether protein kinase C is necessary for the calcium activation of the Na+/H+ exchange in human erythrocytes by studying activation by calcium of erythrocyte Na+/H+ exchange in control cells, in protein kinase C-depleted cells after downregulation of protein kinase C with phorbol-12-myristate-13-acetate and in cells that had been treated beforehand with phorbol-12-myristate-13-acetate with and without the calpain inhibitor E-64d.. Erythrocyte Na+/H+ exchange was measured by determining the initial rates of the influx of Na+ into Na+-depleted, acid loaded cells. The effects of various concentrations (0-1 mmol/l) of CaCl2 and the effects of 1 mmol/l CaCl2 on activation of the intracellular pH and on the external Na+ activation of Na+/H+ exchange were studied. The effects of 1 mmol/l CaCl2 on Na+/H+ exchange in control cells and cells that had been incubated beforehand with and without 1 micromol/l phorbol-12-myristate-13-acetate and with E-64d and 1 micromol/l phorbol-12-myristate-13-acetate for 1, 2, 3 and 24 h were also investigated.. Addition of Ca2+ to a concentration in the range 0-1 mmol/l in the presence of calcimycin resulted in stimulation of Na+/H+ exchange: 1 mmol/l CaCl2 increased (P< 0.001) the erythrocyte Na+/H+ exchange by 74%. Calcium increased the maximum rate for activations by intracellular pH and by external Na+ of Na+/H+ exchange, whereas it did not affect the Michaelis-Menten constants for activation by intracellular H+ and external Na+. However, calcium did not activate the Na+/H+ exchange in protein kinase C downregulated erythrocytes and administration of the calpain inhibitor E-64d could not prevent this inactivation.. Our data indicate that protein kinase C is necessary for the activation by calcium of the erythrocyte Na+/H+ exchange.

Topics: Calcium; Calpain; Cysteine Proteinase Inhibitors; Down-Regulation; Erythrocytes; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Fluid; Ion Transport; Kinetics; Leucine; Male; Protein Kinase C; Sodium; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate

In this study, we report that the cytoplasmic domain of the integrin beta3 subunit is a target for limited proteolysis during apoptosis of human umbilical vein endothelial cells. Calpain inhibitors inhibited the cleavage of the beta3 cytoplasmic domain, indicating that calpain is required. Calpain-mediated proteolysis of fodrin was also detected, indicating that calpain is activated during endothelial cell apoptosis. A phosphatase inhibitor, sodium orthovanadate, inhibited endothelial cell apoptosis and cleavage beta3, suggesting that protein dephosphorylation preceded integrin cleavage in the apoptosis signaling pathway. beta3 cleavage was observed in cells that were viable, suggesting that it is an early event and not the consequence of post-death proteolysis. The extent of beta3 cleavage correlated with a loss in the capacity of cells to reattach to matrix proteins. Loss of reattachment capacity during apoptosis was significantly retarded by a calpain inhibitor. As the beta3 cytoplasmic domain is required for integrin signaling and interaction with the cytoskeleton, our results suggest that cleavage in the beta3 cytoplasmic domain by calpain or a calpain-like protease negatively regulates integrin-mediated adhesion, signaling, and cytoskeleton association.

Topics: Antigens, CD; Apoptosis; Calpain; Carrier Proteins; Cell Adhesion; Cells, Cultured; Cytoskeleton; Endothelium, Vascular; Enzyme Inhibitors; Integrin beta3; Leucine; Microfilament Proteins; Muscle, Smooth, Vascular; Platelet Membrane Glycoproteins; Signal Transduction; Vanadates

Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.

Topics: Alkaline Phosphatase; Animals; Calcium-Binding Proteins; Calpain; Cell Count; Cell Division; Cell Line; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Leucine; Mice; Osteoblasts; Parathyroid Hormone; Rats; Tumor Cells, Cultured

Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca(2+)-dependent effects is unclear. Since cytoskeletal proteins are rapidly degraded by Ca(2+)-dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca(2+)-induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors ethyl(+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butyl carbamoyl]-2 - oxiranecarboxylate (EST) and carbobenzoxyl-valyl-phenylalanyl-H (MDL 28170) to block the Ca2+ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 microM) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 microM) showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 microM) did not block the A23187-induced rise of [Ca2+]i as measured with fura-2. These results suggest that calpains play a role in Ca(2+)-induced pruning of neurites in isolated hippocampal pyramidal neurons.

Topics: Animals; Axons; Calcimycin; Calcium; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Dendrites; Dipeptides; Fetus; Hippocampus; Kinetics; Leucine; Neurites; Pyramidal Cells; Rats; Time Factors

Interleukin 1 alpha (IL-1 alpha) is synthesized as a 33 kDa form and proteolytically processed into a 17 kDa form. Although IL-1 alpha has no signal peptide, it is released from cells. To investigate the relationship between the processing and release of IL-1 alpha, human bladder carcinoma cells (HTB9 5637) which express IL-1 alpha constitutively, were treated with calcium ionophore (A23187). A23187 induced the processing of 33 kDa IL-1 alpha and selectively released processed 17 kDa IL-1 alpha, without any change in the release of 33 kDa IL-1 alpha. When extracellular calcium was chelated by EGTA, or when intracellular calpain was inhibited by the cell-permeable cysteine-protease inhibitor, E64d, the processing of 33 kDa IL-1 alpha was significantly blocked, the release of 33 kDa IL-1 alpha being unchanged. These results indicate that the release of IL-1 alpha was accompanied by the processing of 33 kDa IL-1 alpha.

Topics: Calcimycin; Calcium; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Egtazic Acid; Gene Expression; Humans; Interleukin-1; L-Lactate Dehydrogenase; Leucine; Methionine; Molecular Weight; Protein Processing, Post-Translational; Sulfur Radioisotopes; Tumor Cells, Cultured; Urinary Bladder Neoplasms

E-64d, a membrane-permeable cysteine protease inhibitor, was tested for its ability to inhibit PTH-induced contraction in intact mouse MC-3T3-E1 osteoblastic cells. Incubation of MC-3T3-E1 cells with vehicle (DMSO) or E-64c, a nonpermeant cysteine protease inhibitor, in the presence or in the absence of PTH had no effects on cAMP production or on morphology from 0 to 90 minutes after addition. In contrast, treatment with E-64d markedly attenuated PTH-induced contraction in these cells. These findings suggest that cysteine proteases, such as the calcium-activated neutral proteases (calpains), are involved in PTH-induced osteoblastic contraction. The observation that cysteine protease activity mediates PTH-induced osteoblastic contraction also suggests that endogenous inhibitors, such as calpastatin, may also be present in the osteoblast and play a role in the regulation of stimulus-response coupling in bone. This mechanism may provide another regulatory point at which bone cells may be pharmacologically manipulated in clinical situations characterized by excessive bone resorption.

Topics: Animals; Bone Resorption; Calpain; Cell Line; Cyclic AMP; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Leucine; Mice; Osteoblasts; Parathyroid Hormone

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

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

Intracellular calcium levels play an important role in myofibril disintegration and regeneration of muscle fibers. Earlier studies have shown that the calcium activated protease, calpain, is involved in the removal of Z-discs from myofibrils of striated muscle and the tripeptide-aldehyde, leupeptin, which is an inhibitor of calpain, inhibits this activity. In the present communication, we demonstrate that leupeptin and another calpain inhibitor, E64d, inhibit the fusion of mouse skeletal muscle C2C12 myoblasts to form multinucleated myotubes in tissue culture.

Topics: Animals; Calcium; Calpain; Cell Differentiation; Cell Fusion; Culture Techniques; Leucine; Leupeptins; Mice; Muscles; Protease Inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blood Proteins; Calcimycin; Calcium; Calpain; Enzyme Activation; Erythrocytes; Female; In Vitro Techniques; Leucine; Leupeptins; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Rabbits

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arachidonic Acid; Arachidonic Acids; Calpain; Collagen; Humans; In Vitro Techniques; Leucine; Phosphatidic Acids; Platelet Activation; Prostaglandin Endoperoxides, Synthetic; Protein Kinase C; Serotonin; Tetradecanoylphorbol Acetate; Thrombin

Participation of thiolprotease in platelet activation was investigated. When platelets were treated with EST (L-trans-epoxysuccinyl-leucylamide (3-methyl)butane-ethyl ester, a membrane-permeable thiolprotease inhibitor) for 30 min, thrombin-induced aggregation and secretion were inhibited, and remained so even when the platelets were washed and resuspended in EST-free medium after the pretreatment. The inhibitory action of EST on thrombin-induced platelet aggregation and secretion was both dose-dependent and incubation-time-dependent. The inhibitory action of EST on platelet aggregation and secretion was shown not only in response to thrombin but also to platelet activating factor (PAF). Pretreatment of platelets with 1 mM EST for 30 min inhibited the 65% of calpain (thiolprotease) activity in platelets. The phosphorylation of 40 kDa and 20 kDa proteins of platelets caused by stimulation with thrombin was blocked by the pretreatment with 1 mM EST. Phosphatidylinositol hydrolysis and inositol-1-phosphate production, which appear after stimulation of platelets with thrombin, were also inhibited by the pretreatment with 1 mM EST. The results suggest that EST was incorporated to inside of platelets, and inhibited activation of platelet through inhibition of thiolprotease.

Topics: Adult; Blood Platelets; Calpain; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Inositol Phosphates; Leucine; Phosphatidylinositols; Phosphorylation; Platelet Activating Factor; Platelet Activation; Platelet Aggregation Inhibitors; Serotonin; Thrombin; Type C Phospholipases

E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain.

Topics: Blood Platelets; Calcimycin; Calpain; Cell Membrane Permeability; Cytoskeletal Proteins; Leucine; Microfilament Proteins; Protease Inhibitors; Talin

By employing a cell penetrating thiol protease inhibitor, EST: ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)buty lcarbamoyl]- 2-oxiranecarboxylate, the role of calpain, a major thiol protease in platelets, on 20K protein (myosin light chain) phosphorylation was examined in intact human platelets. EST dose-dependently inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Phosphopeptides mapping revealed the phosphorylation by these agonists was rendered only by the action of myosin light chain kinase (MLCK). However, in TPA (12-O-tetradecanoylphorbol-13-acetate) stimulated platelets, EST did not inhibit 20K phosphorylation which was mediated by the action of C-kinase. [Ca2+]i determined by the use of quin-2 was elevated after the stimulation of thrombin, ionomycin or collagen but not TPA. Thus, it was suggested that calpain enhances MLCK activity on 20K phosphorylation in intact platelets following the stimulation by the agonist which elevates [Ca2+]i.

Topics: Blood Platelets; Calcium; Calpain; Humans; In Vitro Techniques; Leucine; Myosin-Light-Chain Kinase; Myosins; Phosphorylation

E-64 isolated from a culture of Aspergillus japonicus is a specific inhibitor of cysteine proteinases. E-64-c, a synthetic analog of E-64, was effective in model animals of muscular dystrophy only when it was given intraperitoneally and by means of osmotic minipump. It showed no effects due to its low absorbability from intestine when it was administered orally. EST, the ethyl ester of E-64-c, was expected to be readily absorbed through intestinal membrane, since it is more lipophilic than E-64-c. Both EST and E-64-c have a high specificity to cysteine proteinase similar to E-64 but E-64-c was 100 to 1000 times stronger than EST in in vitro cathepsin inhibition. However, EST was stronger than E-64-c in cathepsin inhibition when given orally. The cathepsin B&L activities (whole activities of cathepsins B and L) in the skeletal muscle, heart and liver of hamsters were strongly inhibited soon after oral administration of 100 mg/kg body weight of EST. The inhibition continued for at least 3 h and then disappeared gradually. E-64-c was found in plasma of hamster treated with EST, but unchanged EST was not found. These results suggested that EST was converted to E-64-c, a more active form, during the permeation through intestinal membrane. The conversion of EST to E-64-c was also indicated by the absorption experiment using in situ loop method. EST was thus shown to be useful as an oral drug and expected to be effective in therapeutic trials using model animals.

Topics: Animals; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Endopeptidases; Intestinal Absorption; Leucine; Male; Protease Inhibitors; Rats; Rats, Inbred Strains