Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E(2) release, cytosolic phospholipase (cPLA(2)) steady-state mRNA, cPLA(2) protein levels, cPLA(2) enzyme activity, and cPLA(2) phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E(2) release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA(2) activity at 1 h, and with a significant increase in cPLA(2) Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA(2) phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA(2) phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA(2) activity. Finally, cells stably transfected with an antisense cPLA(2) vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA(2) activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.

Oxidative stress is considered to be an important pathogenic event in ischemia-reperfusion injury, leading to apoptosis or necrosis. We show acute cytotoxicity upon exposure to hydrogen peroxide (H(2)O(2)) in BEAS-2B cells and A549 cells. Single-cell gel electrophoresis showed formation of large comet tails from DNA upon oxidant exposure suggestive of DNA damage. The ATP content of the cells decreased upon exposure to H(2)O(2). Preincubation with 3-aminobenzamide (3-ABA), an inhibitor of poly (ADP-ribosyl) polymerase (PARP), prevented the cytotoxicity. The decrease in the ATP content of the cells was also prevented by 3-ABA. Increase in PARP activity was further confirmed by measuring incorporation of [(32)P]-NAD into nuclear proteins in presence of the cell extracts. Markers of apoptosis were not seen in cells treated with H(2)O(2) with or without 3-ABA pretreatment. These studies suggest that DNA damage is one of the primary reasons for oxidant-induced cell death and that PARP plays an important role in cell death due to its consumption of ATP. Further elaboration of this and other pathways that consume ATP may help prevent oxidant-mediated acute lung injury.

The effect of nitric oxide on p11 expression was studied in an immortalized human bronchial epithelial cell line (BEAS-2B cells). Three nitric oxide donors were used: spermine NONOate (SP), (؎)-S-nitroso-N-acetylpenicillamine (SNAP), and S-nitrosoglutathione (SNOG). All three nitric oxide donors had similar effects resulting in dose-dependent and time-dependent accumulation of p11 protein and an increase of steady-state p11 mRNA. Studies using a reporter gene containing the region from ؊1499 to ؉89 of the p11 promoter demonstrated an increase in transcriptional activity after stimulation with NO donors for 4 h. These effects were abolished at the promoter and protein level using protein kinase G inhibitors (KT5823 and R p -8-pCPT-cGMPS). Incubation of transfected cells with a cell permeable cGMP analogue (8-Br-cGMP) resulted in a doserelated increase of promoter activity. An electrophoretic mobility shift assay of nuclear proteins extracted from BEAS-2B cells identified an AP-1 site located at ؊82 to ؊77 of the p11 promoter region as an NO-and cGMP-dependent response element. These data were confirmed using a c-jun dominant negative mutant vector and a c-jun expression plasmid. Therefore, we conclude that nitric oxide-induced p11 expression in human bronchial epithelial cells is mediated at least in part through increased binding of activator protein one to the p11 promoter.S-100 proteins are a family of proteins first described by Moore et al. (1) who initially characterized a group of low molecular weight (10,000-12,000) acidic proteins in neural tissue. The S-100 group consists of calcium-binding proteins that are expressed in a cell type-dependent fashion. There are at least 13 members, including S-100a, S-100b, and p11 (calpactin light chain). While p11 is a member of the S-100 family, it does not have the ability to bind Ca ϩ2 ions due to crucial amino acid deletions and substitutions in the two EF-hand loops of the protein (2). p11 binds to and inhibits the phosphorylation of annexin II, and binds to the carboxyl region and inhibits cytosolic phospholipase A 2 activity (3). p11-annexin II tetramer is also reported to be a binding protein for cathepsin B, facilitating tumor invasion and metastasis.Nitric oxide is a lipophilic, short-lived, highly reactive free radical, pluripotent molecule involved in the regulation of blood pressure, neurotransmisson, immune function, arachidonic acid metabolism, cell migration, learning and memory, hormone release, cell differentiation, and cell migration (4). Moreover, nitric oxide also plays a role as a messenger in a number of diseases including septic shock, stroke, diabetes mellitus, and rheumatoid arthritis (5). The main source of NO in humans is nitric oxide synthase (present in at least three isoforms), which catalyzes the oxidation of L-arginine to L-citrulline and NO (6). Nitric oxide influences the expression of many genes including cyclooxygenase-2 (7), tumor necrosis factor-␣ (8), interleukin-8 (9), and interleukin-6 (10). Nitric oxide activates soluble, N...

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