Nitric oxide is important in the regulation of mitochondrial function, cell signaling, and gene expression. To elucidate the role of endogenous nitric oxide in the function of airway epithelial cells, we used the compound 1H-imidazol-1-yloxy, 2-(4-carboxyphenyl)-4, 5-dihydro-4, 4, 5, 5-tetramethyl, -3-oxide (PTIO), a hydrophilic, negatively charged nitric oxide trap, to scavenge nitric oxide from rat lung epithelial (RLE) and rat pleural mesothelial (RPM) cells and determine the elicitation of cell cycle alterations, apoptosis, and oxidative stress. The reaction of nitric oxide with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of nitric oxide formation. RLE or RPM cells were exposed to PTIO and supernatants collected to assess the ESR signal of the PTI product. ESR spectroscopy revealed the production of nitric oxide in unexposed RLE or RPM cells. The ESR signal amplitude increased over a period from 1 h to 24 h of exposure, indicating the scavenging of nitric oxide by PTIO. Cell cycle analyses revealed 2- to 3-fold increases in S and G2/M phases after exposure to 100-200 micromole PTIO as well as increases in the fraction of cells undergoing apoptosis. PTIO reacted with nitric oxide to generate PTI prior to addition to cells failed to elicit cell cycle perturbation or apoptosis. The guanylyl cyclase inhibitor, ODQ, mimicked the effects of PTIO, whereas 8-bromo-cyclicGMP ameliorated PTIO-induced apoptosis, suggesting that cGMP dependent pathways are involved in these cell cycle perturbations. Incubation with dichlorofluorescein-diacetate (DCF) revealed oxidative stress in PTIO- but not PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous nitric oxide induces oxidative stress, apoptosis, and cell cycle perturbations. Our findings illustrate the importance of endogenous nitric oxide in the control of cell cycle progression and survival of pulmonary and pleu ral cells and that a critical balance between nitric oxide and superoxide may be necessary for these physiological events.