The role of NOX2 and "novel oxidases" in airway chemoreceptor O(2) sensing.
In pulmonary neuroepithelial bodies (NEB), presumed airway chemoreceptors, classical NADPH oxidase (gp91 phox, NOX2) is co-expressed with O(2) sensitive K(+) channels (K(+)O(2)) and functions as an O(2) sensor. Here we examined related NADPH oxidase homologues "novel oxidases "(NOX 1, 3&4) and their possible involvement in O(2) sensing. For immunolocalization we used specific antibodies against various NADPH components and K(+) (O(2)) subunits to label NEB in rat /rabbit lung and NEB related H146 tumor cell line. For gene expression profiling of NEB cells microdissected from human lung, and H146 cells, we used custom MultiGene-12TM RT-PCR array that included NADPH oxidase components and homologues /accessory proteins (NOX1-4, phox-p22, p40, p47, p67, Rac1, NOXO1 and NOXA1) and K(+)O(2) channels (Kv -1.2, 1.5, 2.1, 3.1, 3.3, 3.4, 4.2, 4.3;TASK1-3). In rat lung, NOX2, NOX4, p22phox, Kv3.3 (and Kv3.4 in rabbit) and TASK1 localized to the apical plasma membrane of NEB cells, and membrane or sub-membrane regions in H146 cells. NEB and H146 cells expressed all NOX proteins except NOX3, as well as all K(+)O(2) channels, except Kv1.5 and Kv4.3. Co-immunoprecipitation using Western blot multicolor Quantum dot labeling showed NOX2 molecular complexes with Kv but not with TASK, while NOX4 associated with TASK1 but not with Kv channel proteins. Hypoxia -induced serotonin release was inhibited in H 146 cells by siRNA to NOX2, while siRNA to NOX4 had only a partial effect, implicating NOX 2 as the predominant NEB cell O(2) sensor. Present findings support NEB cell specific plasma membrane model of O(2) sensing, and suggest unique NOX/K(+)O(2) channel combinations for diverse physiological NEB functions.
Cutz
Ernest
E
Division of Pathology, Department of Pediatric Laboratory Medicine, The Research Institute, The Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada. ernest.cutz@sickkids.ca
Pan
Jie
J
Yeger
Herman
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000939:Epitopes / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D007150:Immunohistochemistry; D007223:Infant; D008168:Lung / Q000166:cytology*; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046568:Neuroepithelial Bodies / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012701:Serotonin / Q000378:metabolism
D000939:Epitopes; D008562:Membrane Glycoproteins; D015221:Potassium Channels; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012701:Serotonin; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D010100:Oxygen