Depletion of nitric oxide causes cell cycle alterations, apoptosis, and oxidative stress in pulmonary cells

Nitric oxide (NO .) is important in the regulation of mitochondrial functio n, cell signaling, and gene expression. To elucidate how endogenous NO . re gulates the function of airway epithelial cells, we used carboxy-PTIO, a hy drophilic, negatively charged NO trap, to scavenge NO . from rat lung epith elial (RLE) and rat pleural mesothelial (RPM) cells and to determine the el icitation of cell cycle alterations, apoptosis, and oxidative stress. The r eaction of NO . with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of NO . format ion. ESR spectroscopy revealed the production of NO . in RLE or RPM cells o ver a period from 1 to 24 h of exposure, indicating scavenging of NO . by P TIO. Cycle analyses in confluent RLE or RPM cells revealed two- to threefol d increases in S and G(2)/M phases after exposure to 100-200 mu M PTIO as w ell as increases in the fraction of cells undergoing apoptosis. Direct addi tion of PTI to cells failed to elicit cell cycle perturbations or apoptosis . The guanylyl cyclase inhibitor ODQ mimicked the effects of PTIO. 8-Bromo- cGMP but not 8-bromo-cAMP ameliorated the PTIO- or ODQ-mediated cell cycle perturbations and apoptosis, suggesting that cGMP-dependent pathways are in volved in these cell cycle perturbations. Treatment of log-phase cells with PTIO resulted in more dramatic cell cycle perturbations compared with cell s treated at confluence. Assessment of 5-bromo-2'-deoxyuridine incorporatio n to measure DNA synthesis demonstrated decreases in PTIO-treated compared with sham cells in addition to a cell cycle arrest in late S or G(2)/M phas e. Last, incubation with dichlorofluorescin diacetate revealed oxidative st ress in PTIO- but not in PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous NO . induces oxidative stress, cell cycle perturba tions, and apoptosis. Our findings illustrate the importance of endogenous NO . in the control of cell cycle progression and survival of pulmonary and pleural cells and that a critical balance between NO . and superoxide may be necessary for these physiological events.