Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia

Abstract

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl− channel controlled in part by the adenosine A2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y2 receptor (P2Y2R) that activates Ca2+-regulated Cl− channels (CaCC/TMEM16) and inhibits Na+ absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10− (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.

Enhancement of Mast Cell Degranulation Mediated by Purinergic Receptor Activation and PI3K(δ)

“…24 Recent studies have emphasized the role of ectonucleotidases in the magnitude of ATP's effects in pulmonary and other organ disorders. 52,53 It seems that many physiological factors interact to either maintain or degrade extracellular ATP in vivo, depending on localized physiological conditions such as pH. It is plausible that data obtained in vitro may be optimized to achieve therapeutic advantages in clinical settings.…”

Airway Epithelial Nucleotide Release Contributes to Mucociliary Clearance

“…The recent identification of the polyoxometalate POM-5 as a potent and effective inhibitor of ATPase activities in airway epithelial cells and lung secretions [23] provided a useful tool to test this hypothesis. As we have recently shown, administration of POM-5 to CF HBE cells results in sustained, increased steady-state ATP levels in airway surfaces and enhanced ASL volume production [23].…”

“…Due to the CFTR defect, the A 2B R/cyclic AMP/CFTR mechanism is impaired in CF and, likely, in cigarette smoke-induced CB. Intriguingly, due to hydrolysis, ATP steady state levels are suboptimal to promote P2Y 2 R-mediated fluid secretion and, therefore, the CFTR-independent mechanism of airway hydration downstream of P2Y 2 R does not optimally hydrate CFTR-deficient epithelia [ 23 ].…”

“…Notably, ATP release increased in HBE cells exposed to inflammatory stresses relevant to CF [ 27 ]. Furthermore, total nucleotide levels, in particular ADP and AMP, were found markedly elevated in CF lung secretions while ATP levels were reduced concomitantly with increased ATPase activity [ 23 ]. ATP concentrations also were reduced in CB lung secretions due to increased ATP hydrolysis activity [ 31 ].…”

“…ATP concentrations also were reduced in CB lung secretions due to increased ATP hydrolysis activity [ 31 ]. These observations suggest that mucus dehydration and inflammation in CF and CB airways are associated with increased ATP metabolism leading to reduced ASL ATP levels [ 23 , 31 ]. Therefore, inhibition of ATP hydrolysis in CF and CB could be an approach to increase steady-state ATP levels on airway surfaces to restore airway hydration via P2Y 2 R-promoted activation of Cl − secretion and inhibition of Na + absorption.…”