Concurrent production of reactive oxygen and nitrogen species by airway epithelial cells in vitro.
Intracellularly generated reactive species of both oxygen (ROS) and nitrogen (RNS) have been implicated in signaling responses in airway epithelial cells, but these radicals have not been measured directly in such cells. In this study, intracellular production of both ROS and RNS were measured in the same cell lysates of guinea pig tracheal epithelial (GPTE) cells maintained in primary culture. ROS and RNS were quantified under basal (constitutive) conditions and in response to different stimuli: LPS and TNFalpha [activators of inducible nitric oxide synthase (iNOS)]; several activators of calcium-dependent cNOS (ATP, bradykinin, ionophore A23187, and thapsigargin); and exogenous oxidant stress generated by addition of xanthine oxidase to purine (p + XO). Studies with LPS and TNFalpha also were performed using the murine macrophage cell line, RAW 264.7, as a positive control. Intracellular oxidant production was detected from oxidation of dihydrorhodamine to rhodamine. NOx was quantified by either chemiluminescent or fluorescent detection. NOS activity was measured as citrulline production from arginine. Basal production of oxidants by GPTE cells (0.08 + 0.00 nmol rhodamine) was less than 10% that of RAW.267 cells (0.91 + 0.03 nmol rhodamine). TNFalpha and LPS significantly increased intracellular oxidant production in GPTE cells, as did p + XO, but none of the cNOS activators affected production of oxidants in these cells. Concentrations of NO2 after 4 h in unstimulated RAW 264.7 and GPTE cells were similar and comprised 63% of total NOx in GPTE and 62% in RAW cells. TNFalpha and LPS both increased NO2 in GPTE cells, but none of the Ca++-mobilizing agents nor p + XO significantly affected intracellular RNS. The results suggest both ROS and RNS can be measured in the same lysates from airway epithelial cells, and that both ROS and RNS are produced in these cells in response to different stimuli.
Rochelle
L G
LG
Cystic Fibrosis/Pulmonary Research Center, University of North Carolina, Chapel Hill, USA.
Fischer
B M
BM
Adler
K B
KB
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D005609:Free Radicals; D006168:Guinea Pigs; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009584:Nitrogen / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014132:Trachea / Q000166:cytology / Q000378:metabolism*
D005609:Free Radicals; D017382:Reactive Oxygen Species; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse; D009584:Nitrogen