benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Respiratory-Distress-Syndrome

Amiodarone is an effective class III antiarrhythmic drug, however, the pulmonary toxicity is one of the most life-threatening complications of its use. The present study was designed to determine the mechanisms underlying pulmonary toxicity of amiodarone. In cultured human lung epithelial cells A549, amiodarone caused cell injury characterized by mitochondrial membrane depolarization, ATP depletion, enhanced propidium iodide (PI) uptake and increase in the number of Annexin-V positive cells, although the population of PI-stained cells appeared earlier and was not identical to that of Annexin-V stained cells, suggesting that the apoptosis and necrosis appeared in different cells. The apoptosis was accompanied with the activation of caspase-2, -3 and -8 but not caspase-9, and reversed by these caspase inhibitors. However, the caspase inhibitors had no influence on mitochondrial membrane potential or PI uptake after exposure of A549 cells to amiodarone. In contrast, mitochondrial cofactors such as L-carnitine and acetyl-L: -carnitine attenuated mitochondrial membrane depolarization, abrogated cellular ATP depletion and reversed PI uptake without affecting Annexin-V positive cells. These finding suggest that different intracellular events operate to cause apoptosis and necrosis after exposure of pulmonary epithelial cells to amiodarone.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Amiodarone; Annexin A5; Apoptosis; Carnitine; Caspase Inhibitors; Cell Survival; Cells, Cultured; Drug Therapy, Combination; Epithelial Cells; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Propidium; Pulmonary Alveoli; Respiratory Distress Syndrome; Tetrazolium Salts

Pseudomonas aeruginosa-induced lung injury is characterized not only by the alteration in lung fluid movement but also by apoptosis of lung epithelial and endothelial cells. We studied whether inhibition of apoptosis using a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), would affect lung fluid balance in rat P. aeruginosa pneumonia.. Z-VAD.fmk (3 mg/kg) was administered intravenously simultaneously with P. aeruginosa intratracheal instillation (0.5 ml/kg, 2 x 10(9) CFU/ml). Apoptosis was evaluated with the TUNEL technique, cytoplasmic oligonucleosome assay, and caspase 3 activation. To evaluate lung permeability, extravascular plasma equivalent (EPE) and lung wet to dry weight ratio (W/D) were measured 4 h after intratracheal instillation of P. aeruginosa.. We found an increase of lung apoptosis 4 h after P. aeruginosa instillation: cytoplasmic oligonucleosome assay increased from 3.17+/-0.78 to 26.82+/-4.67 ODx1000/mg of proteins/ml, Z-VAD.fmk administration decreased this parameter to 10.3+/-2.98 ODx1000/mg of proteins/ml. Caspase 3 levels followed the same pattern. Apoptosis involved both epithelial cells and endothelial cells. Endothelial permeability was increased after Pseudomonas instillation: W/D increased from 3.75+/-0.28 in the Co group to 4.42+/-0.23 in the Pn group; EPE was also higher in the Pn group compared with the Co group (0.125+/-0.04 and 0.002+/-0.01 ml, respectively). Both of these parameters were improved after Z-VAD.fmk administration; W/D decreased to 3.36+/-0.25 and EPE to 0.02+/-0.02 ml.. Apoptosis occurs in the early phase of P. aeruginosa pneumonia. Administration of Z-VAD.fmk significantly decreases DNA fragmentation and caspase 3 levels. This is associated with an improvement of endothelial permeability and lung fluid balance.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Capillary Permeability; Endothelium, Vascular; Pseudomonas Infections; Pulmonary Alveoli; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Statistics, Nonparametric

Earlier work in this laboratory showed that noradrenaline (NA) induces apoptosis in primary cultures of alveolar epithelial cells (AECs). Apoptosis of alveolar epithelial cells may promote the collapse of lung barrier function. On this basis we hypothesized that exogenous NA, administered by intratracheal (I.T.) instillation, might induce AEC apoptosis in vivo followed by acute lung injury. Delivery of NA (10 microM) I.T. into male Wistar rats increased labelling of both fragmented DNA, measured by in situ end labelling (ISEL), and the active form of caspase 3 (anti-Casp3) 6 and 20 h after administration (P < 0.05), but instillation of the vehicle alone (PBS) had no effect. Both ISEL and anti-Casp3 labelling were attenuated by concurrent I.T. delivery of the broad-spectrum caspase inhibitor ZVADfmk. After 6 h, most ISEL- and Casp3-positive cells were located in the surfaces of alveolar walls, but after 20 h more were found in alveolar spaces (P < 0.05). Instillation of NA also increased the bronchoalveolar lavage (BAL) content of fluorescent albumin (BODIPY-alb), which had previously been injected intravenously; the increase was reversed by concurrent ZVADfmk administration. These data suggest that NA-induced apoptosis of AECs in vivo is sufficient to invoke transient collapse of AEC barrier function that is rapidly repaired.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bronchoalveolar Lavage; Caspase 3; Caspase Inhibitors; Caspases; DNA Damage; Epithelial Cells; Male; Norepinephrine; Pulmonary Alveoli; Rats; Rats, Wistar; Respiratory Distress Syndrome

Tumor necrosis factor (TNF)-alpha is released in acute inflammatory lung syndromes linked to the extensive vascular dysfunction associated with increased permeability and endothelial cell apoptosis. TNF-alpha induced significant decreases in transcellular electrical resistance across pulmonary endothelial cell monolayers, reflecting vascular barrier dysfunction (beginning at 4 h and persisting for 48 h). TNF-alpha also triggered endothelial cell apoptosis beginning at 4 h, which was attenuated by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Exploring the involvement of the actomyosin cytoskeleton in these important endothelial cell responses, we determined that TNF-alpha significantly increased myosin light chain (MLC) phosphorylation, with prominent stress fiber and paracellular gap formation, which paralleled the onset of decreases in transcellular electrical resistance and enhanced apoptosis. Reductions in MLC phosphorylation by the inhibition of either MLC kinase (ML-7, cholera toxin) or Rho kinase (Y-27632) dramatically attenuated TNF-alpha-induced stress fiber formation, indexes of apoptosis, and caspase-8 activity but not TNF-alpha-induced barrier dysfunction. These studies indicate a central role for the endothelial cell cytoskeleton in TNF-alpha-mediated apoptosis, whereas TNF-alpha-induced vascular permeability appears to evolve independently of contractile tension generation.

Topics: Actins; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Capillary Permeability; Caspase Inhibitors; Cattle; Cells, Cultured; Cytoskeleton; Electric Impedance; Endothelium, Vascular; Enzyme Inhibitors; Myosin Light Chains; Myosin-Light-Chain Kinase; Phosphorylation; Pulmonary Artery; Respiratory Distress Syndrome; rho GTP-Binding Proteins; Stress Fibers; Tumor Necrosis Factor-alpha