real-time-semi-automated-fluorescent-measurement-airway-surface

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Newcastle University

Transepithelial bicarbonate secretion by human airway submucosal glands and surface epithelial cells is crucial to maintain the pH-sensitive innate defence mechanisms of the lung. cAMP agonists stimulate HCO secretion via coordinated increases in basolateral HCO influx and accumulation, as well as CFTR-dependent HCO efflux at the luminal membrane of airway epithelial cells. Here, we investigated the regulation of a basolateral located, DIDS-sensitive, Cl/HCO exchanger, anion exchanger 2 (AE2; SLC4A2) which is postulated to act as an acid loader, and therefore potential regulator of HCO secretion, in human airway epithelial cells. Using intracellular pH measurements performed on Calu-3 cells, we demonstrate that the activity of the basolateral Cl/HCO exchanger was significantly downregulated by cAMP agonists, via a PKA-independent mechanism and also required Ca and calmodulin under resting conditions. AE2 contains potential phosphorylation sites by a calmodulin substrate, protein kinase CK2, and we demonstrated that AE2 activity was reduced in the presence of CK2 inhibition. Moreover, CK2 inhibition abolished the activity of AE2 in primary human nasal epithelia. Studies performed on mouse AE2 transfected into HEK-293T cells confirmed almost identical Ca/calmodulin and CK2 regulation to that observed in Calu-3 and primary human nasal cells. Furthermore, mouse AE2 activity was reduced by genetic knockout of CK2, an effect which was rescued by exogenous CK2 expression. Together, these findings are the first to demonstrate that CK2 is a key regulator of Cl-dependent HCO export at the serosal membrane of human airway epithelial cells.

CK2 is a key regulator of SLC4A2-mediated Cl/HCO exchange in human airway epithelia.

Lung health relies on effective mucociliary clearance and innate immune defence mechanisms. In cystic fibrosis (CF), an imbalance in ion transport due to an absence of chloride ion secretion, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and a concomitant sodium hyperabsorption, caused by dyregulation of the epithelial sodium channel (ENaC), results in mucus stasis which predisposes the lungs to cycles of chronic infection and inflammation leading to lung function decline. An increased understanding of CFTR structure and function has provided opportunity for the development of a number of novel modulators targeting mutant CFTR however, it is important to also consider other ion channels and transporters present in the airways as putative targets for drug development. In this review, we discuss recent advances in CFTR biology which will contribute to further drug discovery in the field. We also examine developments to inhibit the epithelial sodium channel (ENaC) and potentially activate alternative chloride channels and transporters as a multi-tracked strategy to hydrate CF airways and restore normal mucociliary clearance mechanisms in a manner independent of CFTR mutation.

Ion channels as targets to treat cystic fibrosis lung disease.

Respiratory failure, driven by airways mucus obstruction, chronic inflammation and bacterial infections, is the main cause of mortality and morbidity in people with cystic fibrosis (CF) due to defects in the Cl and  transport activity of the CF Transmembrane conductance Regulator (CFTR). Most recent pre-clinical and clinical studies have focused on restoring CFTR function by enhancing its trafficking or transport activity and show promising results. However, there are a significant number of patients that will not benefit from these CFTR-targeted therapies and it is therefore important to identify new non-CFTR targets that will restore lung function, by-passing CFTR dysfunction. The H/K-ATPase, ATP12A, has recently been identified as a potential novel target for CF therapies, since its acute inhibition by ouabain was shown to help restore mucus viscosity, mucociliary transport, and antimicrobial activity using  CF airway models, and this effect was linked to an increase in the pH of the airway surface liquid (ASL). Here, we have evaluated the potential therapeutic use of ouabain by investigating the effect of chronically treating fully differentiated CF primary human airway epithelial cells (hAECs) with ouabain, under thin film conditions, resembling the  situation. Our results show that although chronic treatment increased ASL pH, this correlated with a deleterious effect on epithelial integrity as assessed by LDH release, transepithelial electrical resistance, fluorescein flux, and ion transport. Since ATP12A shares approximately 65% identity with the gastric H/K-ATPase (ATP4A), we investigated the potential of using clinically approved ATP4A proton pump inhibitors (PPIs) for their ability to restore ASL pH in CF hAECs. We show that, despite not expressing ATP4A transcripts, acute exposure to the PPI esomeprezole, produced changes in intracellular pH that were consistent with the inhibition of H secretion, but this response was independent of ATP12A. More importantly, chronic exposure of CF hAECs to esomeprazole alkalinized the ASL without disrupting the epithelial barrier integrity, but this increase in ASL pH was consistent with a decrease in mRNA expression of . We conclude that PPIs may offer a new approach to restore ASL pH in CF airways, which is independent of CFTR.

Article	Year
CK2 is a key regulator of SLC4A2-mediated Cl/HCO exchange in human airway epithelia. Pflugers Archiv : European journal of physiology\| PubMed ID: 28455748	2017
Ion channels as targets to treat cystic fibrosis lung disease. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 29102290	2018
Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells. Frontiers in pharmacology\| PubMed ID: 30618754	2018

Vinciane Saint-Criq

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