benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Hyperoxia

Therapy with high oxygen concentrations (hyperoxia) is often necessary to treat patients with respiratory failure. However, hyperoxia may exacerbate the development of acute lung injury, perhaps by increasing lung epithelial cell death. Therefore, interrupting lung epithelial cell death is an important protective and therapeutic strategy. In the present study, hyperoxia (95% O(2)) results in murine lung epithelium cell death by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end labeling, and Annexin V-fluorescein isothiocyanate flow cytometry assay. We show that hyperoxia increases superoxide production, as assessed by nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity and flow cytometric assay, and increases phospho-extracellular signal-regulated kinase (ERK)1/2 by Western blot analysis. These processes are inhibited by a reactive oxygen species inhibitor, diphenylene iodonium (DPI), and by an inhibitor of the mitogen-activated protein (MAP) or ERK kinase (MEK)/ERK1/2 pathway, PD98059. ERK1/2 activation in hyperoxia is also inhibited by DPI. Hyperoxia-induced cell death is associated with cytochrome c release, subsequent caspase 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, which can all be suppressed by DPI and PD98059. However, the broad caspase inhibitor z-VAD-FMK protects cells from death without affecting superoxide generation and ERK1/2 activation. Taken together, our data suggest that hyperoxia, by virtue of activating NADPH oxidase, generates reactive oxygen species (ROS), which mediates cell death of lung epithelium via ERK1/2 MAPK activation, and functions upstream of caspase activation in lung epithelial cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspases; Cell Death; Cell Line, Transformed; Cysteine Proteinase Inhibitors; Electron Transport Complex IV; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Flavonoids; Hyperoxia; Lung; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; NADPH Oxidases; Onium Compounds; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Signal Transduction

Mice exposed to 100% O2 die after 3 or 4 d with diffuse alveolar damage and alveolar edema. Extensive cell death is evident by electron microscopy in the alveolar septa, affecting both endothelial and epithelial cells. The damaged cells show features of both apoptosis (condensation and margination of chromatin) and necrosis (disruption of the plasma membrane). The electrophoretic pattern of lung DNA indicates both internucleosomal fragmentation, characteristic of apoptosis, and overall degradation, characteristic of necrosis. Hyperoxia induces a marked increase in RNA or protein levels of p53, bax, bcl-x, and Fas, which are known to be expressed in certain types of apoptosis. However, we did not detect an increased activity of proteases belonging to the apoptosis "executioner" machinery, such as CPP32 (caspase 3), ICE (caspase 1), or cathepsin D. Furthermore, administration of an ICE-like protease inhibitor did not significantly enhance the resistance to oxygen. Additionally, neither p53-deficient mice nor lpr mice (Fas null) manifested an increased resistance to hyperoxia-induced lung damage. These results show that both necrosis and apoptosis contribute to cell death during hyperoxia. Multiple apoptotic pathways seem to be involved in this, and an antiapoptotic strategy does not attenuate alveolar damage.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cathepsin D; Cysteine Proteinase Inhibitors; Fas Ligand Protein; fas Receptor; Gene Expression; Hyperoxia; In Situ Nick-End Labeling; Lung Diseases; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Oxygen; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pulmonary Alveoli; RNA, Messenger; Tumor Suppressor Protein p53