Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Air-way epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H2O2), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the cur-rent study we examine the biological consequence of exposure of human air-way epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO-), apoptosis was observed. Similarly pure ONOO--induced apoptosis, and ONOO--induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO--induced apoptosis, but did prevent H2O2-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H2O2 specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO- exposure did not affect neutral sphingomyelinase activity; rather, ONOO- specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H2O2-induced apoptosis, but had no effect on ONOO--induced apoptosis. on the other hand, silencing of aSMase markedly impaired ONOO--induced apoptosis, but did not affect H2O2-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury. (C) 2007 Elsevier Inc. All rights reserved.