cathepsin-g and phosphoramidon

High activities of the neutrophil proteases, elastase and cathepsin G, are found in the sputum of patients with cystic fibrosis (CF). Because both proteases have been shown to be potent secretagogues for airway submucosal glands, and because hypersecretion is a characteristic feature of CF, the objective of the present study was to examine whether there is secretagogue activity in CF sputum, and to determine the contribution of neutrophil proteases to the secretagogue activity. Confluent monolayers of cultured bovine tracheal serous cells were pulse-labelled with Na2(35)SO4, incubated with diluted CF sputum supernatants in the presence or absence of different protease inhibitors, and the subsequent release of the radio-labelled macromolecules was measured. CF sputum potently induced secretion concentration-dependently. Addition of the selective neutrophil elastase inhibitor ICI 200,355 inhibited the secretory response to CF sputum supernatant by 89%. Addition of a cathepsin G-inhibitor resulted in further inhibition of the secretory response. Addition of phosphoramidon, a drug known to inhibit Pseudomonas aeruginosa elastase, had no effect. We conclude that CF sputum potently stimulates airway submucosal gland cell secretion. These studies with protease inhibitors suggest that neutrophil proteases account substantially for the secretagogue activity present in CF sputum.

Topics: Adult; Animals; Cathepsin G; Cathepsins; Cattle; Cells, Cultured; Cystic Fibrosis; Exocrine Glands; Glycopeptides; Humans; Leukocyte Elastase; Oligopeptides; Pancreatic Elastase; Protease Inhibitors; Serine Endopeptidases; Sputum; Trachea

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide secreted by endothelial cells. We investigated whether polymorphonuclear neutrophils (PMN) were able to destroy this peptide by enzymatic hydrolysis produced either by the membrane-bound endopeptidase 24.11 or by lysosomal proteinases released in the medium by activated cells. Resting and activated PMN were incubated with 125I-labelled ET-1 and the degradation fragments were analyzed by HPLC. A marked degradation of ET-1 was observed only in the presence of the stimulated cells, leading to the generation of seven radiolabelled peaks. Addition of phosphoramidon had no effect on the appearance of these metabolites, while soybean trypsin inhibitor abolished almost completely the degradation of the peptide, suggesting a role of cathepsin G in ET-1 hydrolysis. Among the purified leukocyte enzymes tested, cathepsin G hydrolyzed 125I-labelled ET-1 at the higher rate and gave rise to two radiolabelled peaks already observed in the presence of activated PMN. Incubation of unlabelled ET-1 with purified cathepsin G allowed to identify a major cleavage site corresponding to the His16-Leu17 bond, leading to the formation of inactive [1-16] fragments and the C-terminal pentapeptide. This mechanism of ET-1 inactivation could play a role in acute inflammatory reaction where PMN adhere to the vascular endothelial cells.

Topics: Cathepsin G; Cathepsins; Chromatography, High Pressure Liquid; Endopeptidases; Endothelins; Glycopeptides; Humans; Hydrolysis; Neprilysin; Neutrophils; Pancreatic Elastase; Serine Endopeptidases; Tetradecanoylphorbol Acetate; Trypsin Inhibitor, Kunitz Soybean