H₂O₂ Stimulates Cystic Fibrosis Transmembrane Conductance Regulator through an Autocrine Prostaglandin Pathway, Using Multidrug-Resistant Protein–4

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H 2 O 2 levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air-liquid interface were estimated, and H 2 O 2 -mediated changes in CFTR activity were examined. In Ussing chambers, H 2 O 2 -induced anion currents were sensitive to the CFTR inhibitors CFTR inh 172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Effective airway clearance relies on the regulation of ciliary beat frequency (CBF), adequate airway surface liquid (ASL) volume, and correct mucus properties. Mucus hydration and thus the viscosity of mucus depend, in part, on the movement of water through the airway epithelium to the mucosal surface via the coordinated activities of ion channels. An important player in this regulation is the cystic fibrosis transmembrane conductance regulator (CFTR) that passes chloride (and other anions) while regulating the epithelial Na 1 channel to balance osmolarity (1). CFTR and CBF are both regulated through increases in cyclic adenosine monophosphate (cAMP) and protein kinase A activity (2-4). Thus, mediators that stimulate adenylyl cyclase are expected to result in improved airway clearance by increasing chloride secretion and CBF.Airway epithelia actively synthesize H 2 O 2 via the nicotinamide adenine dinucleotide phosphate-reduced (NADPH) dual oxidases Duox1 and Duox2, as reviewed by Fischer (5), presumably for the use of the lactoperoxidase host defense against infection (6-9). Duox1 and Duox2 are regulated by cytokines (10-12) and bacterial products (10,13,14) to increase H 2 O 2 synthesis to high levels (10). Stimulation with interferon-g increased H 2 O 2 synthesis levels 30-fold when measured in air-liquid interface (ALI) cultures of normal human airway epithelial cells (10), leading to 10 mM H 2 O 2 in washes used for assays. The experiments presented here address ASL H 2 O 2 and show that it is present at high concentrations.In addition to its role in stimulating lactoperoxidase host defense, H 2 O 2 may also regulate the innate defense related to mucociliary clearance. In the bronchial submucosal adenocarcinoma cell line Calu-3, CFTR activity is increased by exposure of the airway epithelium to exogenous H 2 O 2 (15, 16). In cell lines, the effect of H 2 O 2 on CFTR is reported to occur through increases in transmembrane adenylyl cyclase activity and through the activation of two K 1 channels (15), most likely KCNQ1 and KCNN4, which are located in the basolateral membrane. These channels are believed to provide a driving force for apical Cl 2 release (17, 18). Yet the mechanisms by which H 2 O 2 leads to the activation of the cAMP pathway are not understood. Although both Calu-3 cells (19) and bovine tracheal epithelia (20) have been shown to respond to isop...

Chronic β2AR stimulation limits CFTR activation in human airway epithelia

“…Acute MRP4 blockade did not rescue CFTR activation by forskolin/IBMX in either wtCFTR + (Supplemental Figure 1C) or VX809-corrected F508del-CFTR + (Supplemental Figure 1D) cells pretreated with albuterol. In wtCFTR + cells, MRP4 blockade further decreased CFTR activation; this phenomenon has been previously noted in airway epithelial cells, and may relate to PGE 2 efflux through MRP4 (36). Regardless, our results indicate that MRP4 inhibition was not sufficient to restore CFTR activation following chronic β2AR stimulation.…”

Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production

“…However, similar results were obtained in NHBE cells using H 2 O 2 . Duox2 can create at least a concentration of 150 M H 2 O 2 in the apical fluid above airway cells, and 0.5 mM is a reasonable maximum (16). Therefore, we used 0.5 mM H 2 O 2 for our human cell experiments.…”

“…We have shown that IFN-␥ can increase H 2 O 2 production via increased expression of Duox2, a member of the NADPH oxidase gene family, in ALI 2 cultures (15,16). In addition to H 2 O 2 , Duox also releases cytosolic H ϩ (17), which could contribute to intracellular acidification.…”

The impact of cigarette smoke exposure, and COPD or asthma status on ABC transporter gene expression in human airway epithelial cells

“…Equally convincing of the role of ABC transporters in lung health and disease is the link between genetic loss of function variants of ABCA3 and fatal surfactant deficiency in newborns 12 . In addition, our group has recently confirmed that ABCC4 is expressed in airway epithelial cells 4,5 , is able to transport cAMP and uric acid 5 , and can modulate anti-inflammatory activities of long-acting beta-agonist/glucocorticoid therapies 6 and potentiate CFTR in select patient populations 13 . These three examples represent only a fraction of the 48 transporters that remain to be more extensively studied in the respiratory mucosa for expression patterns and function in lung health and disease.…”

“…A relatively unexplored contributor to respiratory mucosa biology is the ATP Binding Cassette (ABC) family of transporters [3][4][5][6][7] . ABC transporters are conserved in prokaryotes and eukaryotes, with humans expressing 48 transporters divided into 7 classes (ABCA, ABCB, ABCC, ABCD, ABDE, ABCF, and ABCG) (7).…”