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1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H(2)O(2). To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.

Smoking is a major risk factor for endothelial cell injury and subsequent coronary artery disease. Epidemiological studies implicate the phospholipase A2/arachidonic acid cascade in the mechanism by which smoking causes heart disease. However, specific components of cigarette smoke that activate this pathway have not been identified. The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons contained in cigarette smoke on phospholipase A2 (PLA2) activity and apoptosis of human coronary artery endothelial cells. 1-methylanthracene (1-MA), phenanthrene (PA), and benzo(a)pyrene (B(a)P) caused significant release of 3H-arachidonate from endothelial cells. 1-MA and PA, but not B(a)P, also caused significant release of 3H-linoleic acid. Release of fatty acids from membrane phospholipids preceded the onset of apoptosis. 3H-arachidonate release and apoptosis induced by 1-MA, B(a)P, and PA were inhibited by methylarachidonoyl-fluorophosphonate, an inhibitor of Groups IV and VI PLA2s. Bromoenol lactone, an inhibitor of Group VI enzymes, inhibited both 3H-arachidonate release and apoptosis induced by 1-MA and PA, but not B(a)P. MJ33, an inhibitor of the acidic calcium-independent PLA2, attenuated 3H-arachidonate release and apoptosis by PA, but not 1-MA or B(a)P. The presence of Groups IV and VI and the acidic iPLA2 in endothelial cells was demonstrated by reverse transcriptase-polymerase chain reaction and Western analysis. These data suggest that 1-MA, B(a)P and PA induce apoptosis of endothelial cells by a mechanism that involves activation of these three distinct isoforms of PLA2.

1-cys peroxiredoxin (1-cys Prx), the only member of a Prx subfamily that contains a single conserved cysteine residue, is abundant in lung. This bifunctional protein has both glutathione peroxidase and phospholipase A2 activities compatible with a role both in protection against lung oxidant injury and also in lung phospholipid metabolism. Here we studied the developmental expression of 1-cys Prx in rat lungs and hormonal effects on protein expression in human and rat lung cells. There was little change in 1-cys Prx expression during the prenatal period, but a marked increase in expression immediately after birth. Enzymatic (peroxidase and phospholipase) activities increased gradually after birth and reached adult level at 7-14 postnatal days. Expression of the protein was induced in the presence of dexamethasone (Dex) in cultured human and rat lung epithelial cells and also was upregulated in neonatal rat lung in vivo. cAMP treatment had no effect on expression, although there was a modest synergistic effect when combined with Dex in human fetal lung epithelial cells. The increased expression of 1-cys Prx at birth may be important for surfactant phospholipid turnover related to the phopholipase A2 activity of the protein and for antioxidant defense based on its peroxidase function.

Lamellar bodies are the specialized secretory organelles of alveolar type II (ATII) epithelial cells through which the cell packages pulmonary surfactant and regulates its secretion. Surfactant within lamellar bodies is densely packed as circular arrays of lipid membranes and appears to be the product of several trafficking and biosynthetic processes. To elucidate these processes, we reported previously on the generation of a monoclonal antibody (3C9) that recognizes a unique protein of the lamellar body membrane of 180 kDa, which we named LBM180. We report that mass spectrometry of the protein precipitated by this antibody generated a partial sequence that is identical to the ATP-binding cassette protein, ABCA3. Homology analysis of partial sequences suggests that this protein is highly conserved among species. The ABCA3 gene transcript was found in cell lines of human lung origin, in ATII cells of human, rat, and mouse, as well as different tissues of rat, but the highest expression of ABCA3 was observed in ATII cells. Expression of this transcript was at its maximum prior to birth, and hormonal induction of ABCA3 transcript was observed in human fetal lung at the same time as other surfactant protein transcripts were induced, suggesting that ABCA3 is developmentally regulated. Molecular and biochemical studies show that ABCA3 is targeted to vesicle membranes and is found in the limiting membrane of lamellar bodies. Because ABCA3 is a member of a subfamily of ABC transporters that are predominantly known to be involved in the regulation of lipid transport and membrane trafficking, we speculate that this protein may play a key role in lipid organization during the formation of lamellar bodies.

We have shown previously that ischemia in an isolated rat lung that is normally oxygenated by continued ventilation results in lipid and protein oxidation, indicating the generation of reactive oxygen species. By using a variety of biochemical and imaging techniques, we determined that the initial response, which occurs within the first second of ischemia, is partial depolarization of the endothelial cell plasma membrane. This event is followed within several seconds by activation of endothelial NADPH oxidase and generation of superoxide anion at the extracellular surface of the cell membrane where it is dismutated to freely diffusible H2O2. Approximately 15 secs after the onset of ischemia, we detected an elevation of intracellular Ca2+ caused by its release from intracellular stores, followed by Ca2+ influx, possibly through T-type voltage-dependent Ca2+ channels. Increased nitric oxide generation through activation of endothelial nitric oxide synthase is detected after about 45 secs of ischemia. These changes (endothelial membrane depolarization, reactive oxygen species production, elevation of intracellular Ca2+ levels, and nitric oxide generation) were confirmed in isolated endothelial cells that had been adapted to shear stress in vitro. The in vitro model also demonstrates reactive oxygen species-dependent activation of nuclear factor-kappaB and activator protein-1 and that 24 hrs of ischemia results in increased cell division. These results indicate a novel cell-signaling pathway in response to loss of shear stress. The basis for these changes in endothelial function is related to mechanotransduction, i.e., altered shear stress rather than a metabolic response to ischemia. The biological function for the response may be an attempt to restore blood flow through vasodilatation and new capillary formation.