Adaptation to Supraphysiologic Room Air Oxygen Changes the Effects of Nitric Oxide on A549 Lung Cells In Vitro

Abstract

Airway endothelial cells are exposed to some of the highest oxygen levels encountered in vivo (~10% O2), however, these levels are significantly lower than room air (21% O2). The culture and handling of airway cells in room air oxygen incubators can change the redox status of the cells, introducing artifact. Nitric oxide, which can affect HIF‐1a stabilization and reversibly bind cytochrome oxidase, has been shown to play multiple roles in the response of lung to hypoxic injury and inflammation. Here we explore whether pre‐adaptation of the commonly used human lung epithelial cell line A549 to room air oxygen levels changes cellular responsiveness to NO. The null hypothesis was that pre‐adaptation to different oxygen levels would have no effect on responses to NO. Using the Xvivo System to provide full‐time control of oxygen and CO2 levels during cell incubation as well as handling, we adapted A549 cells for growth at 18% supraphysiologic oxygen (standard room air incubator level with humidity and CO2), physiologically relevant oxygen (10%) or pathophysiologic hypoxia (2% O2). We assayed cellular responses to gaseous nitric oxide (NO) exposure in standard plate‐based cytotoxicity and migration assays and assayed local nitric oxide levels in the cell culture media. We found that adaptation to different oxygen levels had a concentration‐dependent effect on the responses of A549 cells to gaseous NO. We concluded that it is critical to consider the oxygen‐adapted state of the cell prior to assay to avoid oxygen artifact.Support or Funding InformationAll support was from BioSpherix, Ltd.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.