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1 Department of Pathology, University of Vermont, Burlington, Vermont 05405; and 2 Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Nitric oxide (NO ·) is important in the regulation of mitochondrial function, cell signaling, and gene expression. To elucidate how endogenous NO · regulates the function of airway epithelial cells, we used carboxy-PTIO, a hydrophilic, negatively charged NO · trap, to scavenge NO · from rat lung epithelial (RLE) and rat pleural mesothelial (RPM) cells and to determine the elicitation of cell cycle alterations, apoptosis, and oxidative stress. The reaction of NO · with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of NO · formation. ESR spectroscopy revealed the production of NO · in RLE or RPM cells over a period from 1 to 24 h of exposure, indicating scavenging of NO · by PTIO. Cycle analyses in confluent RLE or RPM cells revealed two- to threefold increases in S and G2/M phases after exposure to 100-200 µM PTIO as well as increases in the fraction of cells undergoing apoptosis. Direct addition 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 involved in these cell cycle perturbations. Treatment of log-phase cells with PTIO resulted in more dramatic cell cycle perturbations compared with cells treated at confluence. Assessment of 5-bromo-2'-deoxyuridine incorporation to measure DNA synthesis demonstrated decreases in PTIO-treated compared with sham cells in addition to a cell cycle arrest in late S or G2/M phase. Last, incubation with dichlorofluorescin diacetate revealed oxidative stress in PTIO- but not in PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous NO · induces oxidative stress, cell cycle perturbations, 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.
nitronyl nitroxide; epithelial cell; mesothelial cell
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