NADPH oxidase is an O₂ sensor in airway chemoreceptors: Evidence from K⁺ current modulation in wild-type and oxidase-deficient mice

Pulmonary neuroepithelial bodies (NEBs) are presumed airway chemoreceptors that express the putative O₂ sensor protein NADPH oxidase and O₂-sensitive K⁺ channels K⁺(O₂). Although there is a consensus that redox modulation of K⁺(O₂) may be a common O₂-sensing mechanism, the identity of the O₂ sensor and related coupling pathways are still controversial. To test whether NADPH oxidase is the O₂ sensor in NEB cells, we performed patch- clamp experiments on intact NEBs identified by neutral red staining in fresh lung slices from wild-type (WT) and oxidase-deficient (OD) mice. In OD mice, cytochrome b₅₅₈ and oxidase function was disrupted in the gp91(Phox) subunit coding region by insertion of a neomycin phosphotransferase (neo) gene. Expression in NEB cells of neo mRNA, a marker for nonfunctional gp91(phox) was confirmed by nonisotopic in situ hybridization. In WT cells, hypoxia (pO₂ = 15-20 mmHg; 1 mmHg = 133 Pa) caused a reversible inhibition (κ46%) of both Ca²⁺-independent and Ca²⁺dependent K⁺ currents. In contrast, hypoxia had no effect on K⁺ current in OD cells, even though both K⁺ current components were expressed. Diphenylene iodonium (1 μM), an inhibitor of the oxidase, reduced K⁺ current by κ30% in WT cells but had no effect in OD cells. Hydrogen peroxide (H₂O₂; 0.25 mM), a reactive oxygen species generated by functional NADPH oxidase, augmented K⁺ current by >30% in both WT and OD cells; further, in WT cells, H₂O₂ restored K⁺ current amplitude in the presence of diphenylene iodonium. We conclude that NADPH oxidase acts as the O₂ sensor in pulmonary airway chemoreceptors.