Regulated hydrogen peroxide production by Duox in human airway epithelial cells.
Hydrogen peroxide (H(2)O(2)) is found in exhaled breath and is produced by airway epithelia. In addition, H(2)O(2) is a necessary substrate for the airway lactoperoxidase (LPO) anti-infection system. To investigate the source of H(2)O(2) produced by airway epithelia, PCR was used to screen nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in human airway epithelia redifferentiated at the air-liquid interface (ALI) and demonstrated the presence of Duox1 and 2. Western blots of culture extracts indicated strong expression of Duox, and immunohistochemistry of human tracheal sections localized the protein to the apical portion of epithelial cells. Apical H(2)O(2) production was stimulated by 100 microM ATP or 1 microM thapsigargin, but not 100 microM ADP. Diphenyleneiodonium, an NADPH oxidase inhibitor, and dimethylthiourea, a reactive oxygen species scavenger, both inhibited this stimulation. ATP did not stimulate the basolateral H(2)O(2) production by ALI cultures. ATP and thapsigargin increased intracellular Ca(2+) with kinetics similar to increasing H(2)O(2) production, and thus consistent with the expected Ca(2+) sensitivity of Duox. These data suggest that Duox is the major NADPH oxidase expressed in airway epithelia and therefore a contributor of H(2)O(2) production in the airway lumen. In addition, the data suggest that extracellular H(2)O(2) production may be regulated by stimuli that raise intracellular Ca(2+).
Forteza
Radia
R
University of Miami School of Medicine, P.O. Box 016960 (R124), Miami, FL 33101, USA.
Salathe
Matthias
M
Miot
Françoise
F
Forteza
Rosanna
R
Conner
Gregory E
GE
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity; D000074623:Dual Oxidases; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005420:Flavoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D010084:Oxidation-Reduction; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism; D019284:Thapsigargin / Q000378:metabolism; D014132:Trachea / Q000166:cytology / Q000378:metabolism
D005420:Flavoproteins; D010078:Oxazines; D016877:Oxidants; C470430:Amplex Red; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D006735:Horseradish Peroxidase; D019255:NADPH Oxidases; C431513:DUOX1 protein, human; C431515:DUOX2 protein, human; D002118:Calcium