The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non-small cell lung carcinomas. Understanding how cholinergic signaling is upregulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of A5 and B3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogenactivated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC.

Pulmonary neuroepithelial bodies (NEBs) are presumed airway chemoreceptors that express the putative O2 sensor protein NADPH oxidase and O 2-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 O 2 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 oxidasedeficient (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 (Ϸ46%) of both Ca 2؉ -independent and Ca 2؉ -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 M), an inhibitor of the oxidase, reduced K ؉ current by Ϸ30% 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 O 2 sensor in pulmonary airway chemoreceptors.