calpastatin and Acute-Disease

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Topics: Actins; Acute Disease; Animals; Blotting, Western; Cadherins; Calcium-Binding Proteins; Calpain; Ceruletide; Cholecystokinin; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Cytoskeleton; Dipeptides; Enzyme Activation; Female; Gene Expression; Microscopy, Confocal; Microscopy, Electron; Models, Animal; Organ Culture Techniques; Pancreas; Pancreatitis; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrin; Stimulation, Chemical; Time Factors; Vinculin

Surfactant deficiency is an important contributor to the acute respiratory distress syndrome, a disorder that commonly occurs after bacterial sepsis. CTP:phosphocholine cytidylyltransferase (CCTalpha) is the rate-limiting enzyme required for the biosynthesis of dipalmitoylphosphatidylcholine (DPPC), the major phospholipid of surfactant. In this study, a cDNA encoding a novel, calpain-resistant mutant CCTalpha enzyme was delivered intratracheally in mice using a replication-deficient adenovirus 5 CTP:phosphocholine cytidylyltransferase construct (Ad5-CCT(Penta)) in models of bacterial sepsis. Ad5-CCT(Penta) gene transfer produced high-level CCTalpha gene expression, increased alveolar surfactant (DPPC) levels and improved lung surface tension and pressure-volume relationships relative to control mice. Pseudomonas aeruginosa (PA103) decreased DPPC synthesis, in part, via calpain-mediated degradation of CCTalpha. Deleterious effects of Pseudomonas on surfactant were lessened after infection with a mutant strain lacking the type III exotoxin, Exo U. Replication-deficient adenovirus 5 CTP:phosphocholine cytidylyltransferase gene delivery improved lung biophysical properties by optimizing surface activity in this Pseudomonas model of proteinase-mediated lung injury. The studies are the first demonstration of in vivo gene transfer of a lipogenic enzyme resulting in improved lung mechanics. The studies suggest that augmentation of DPPC synthesis via gene delivery of CCTalpha can attenuate impaired lung function in surfactant-deficient states such as bacterial sepsis.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Acute Disease; Adenoviridae; Animals; Calcium-Binding Proteins; Choline-Phosphate Cytidylyltransferase; Genetic Therapy; Genetic Vectors; Lung; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Pseudomonas Infections; Pulmonary Surfactants; Transduction, Genetic

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.

Topics: Acute Disease; Animals; Calcium-Binding Proteins; Calpain; Ceruletide; Dipeptides; Enzyme Activation; Female; Pancreas; Pancreatitis; Rats; Rats, Inbred Lew; Severity of Illness Index

STATEMENT OF FINDINGS: An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinically and followed prospectively for 1 year to determine the clinical significance of a number of rheumatoid arthritis (RA) associated autoantibodies. Serum samples collected at the time of the initial evaluation were tested for rheumatoid factor (RF) and antibodies to Sa (anti-Sa), RA-33, (pro)filaggrin [antifilaggrin antibody (AFA)], cyclic citrullinated peptide (anti-CCP), calpastatin, and keratin [antikeratin antibody (AKA)]. RF had a sensitivity of 66% and a specificity of 87% for RA. Anti-Sa, AFA, and anti-CCP all had a specificity of more than 90%, but a sensitivity of less than 50% for this diagnosis. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses.

Topics: Acute Disease; Adult; Antibody Specificity; Arthritis, Rheumatoid; Autoantibodies; Calcium-Binding Proteins; Citrulline; Coenzyme A Ligases; Cohort Studies; Epitopes; Female; Filaggrin Proteins; Histocompatibility Testing; Humans; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Peptides, Cyclic; Predictive Value of Tests; Proteins; Rheumatoid Factor; Seroepidemiologic Studies; Synovitis