Prostaglandin E2‐Stimulated Cl− Secretion in Airway Epithelium Involves Protein Kinase A and Calcium Crosstalk

Abstract

BackgroundMucociliary clearance is an integral defense mechanism against pulmonary infections and is impaired in Cystic Fibrosis (CF) airways. Prostaglandin E2 (PGE2) is abundant in infected airways and stimulates transepithelial Cl− secretion and mucociliary clearance, both of which are impaired in CF. Previously, we found that PGE2‐stimulated Cl− secretion was cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent in bronchial epithelial cells, but in submucosal gland cells utilized both CFTR and Ca2+‐activated Cl− channels (CaCC).AimTo determine the signaling mechanism(s) whereby PGE2 stimulates airway Cl− secretion in order to identify ways to circumvent the mucociliary defect in CF.MethodsHuman bronchial epithelial cell cultures (CFBE41o‐ with wildtype CFTR) were used for all experiments. Clsecretion was measured by short‐circuit current (Isc) with cell monolayers mounted in Ussing chambers and exposed to a serosal to mucosal Cl− gradient. These conditions minimize contribution from basolateral K+ channels and Isc reflects primarily Cl− transport. EP receptor expression was determined by qPCR.ResultsIn bronchial epithelial cell cultures, qPCR showed that EP4 receptor expression was 148% greater than EP3 receptor expression. CAY10598 (1 μM), a selective EP4 agonist, stimulated Cl− secretion to a similar magnitude as PGE2 (Cay: 85.5 +/− 8.6 μA/cm2 (n=4) vs. PGE2: 81.6 +/− 10.2 μA/cm2 (n=17); mean +/− SE). In contrast, sulprostone, an EP3 agonist, was less effective at 1 or 10 μM (5.6+/− 2.0 μA/cm2 (n=6) and 14.5+/− 7.3 μA/cm2 (n=2), respectively). Pre‐treatment with H‐89 (10 μM), a protein kinase A (PKA) inhibitor, decreased PGE2‐stimulated Cl− secretion by 74% (n=9). Similarly, pretreatment with BAPTA‐AM (100 μM), a Ca2+ chelator, inhibited PGE2‐stimulated Cl− secretion by 71% (n=7). Simultaneous pre‐treatment with H‐89 and BAPTA‐AM was additive and completely abolished PGE2‐stimulated Cl− secretion (94% inhibition, n=3). Additionally, wortmannin (1μM), a phosphoinositide 3‐kinase (PI3K) inhibitor, decreased PGE2‐stimulated Clsecretion by 56% (n=4).Summary and ConclusionsPGE2‐stimulated Cl− secretion in human bronchial epithelial cells predominantly occurs via EP4 receptors. While utilizing classic EP4 receptor signaling (PKA and PI3K), there is also substantial crosstalk with intracellular Ca2+. Interestingly, the latter does not activate CaCC, but rather CFTR. Ongoing experiments are aimed at further delineating the intracellular signaling mechanisms involved in PGE2‐stimulated Cl− secretion in bronchial epithelial cells, as well as comparing these mechanisms to PGE2‐stimulated Cl− secretion in submucosal gland cells. Through this we hope to identify potential ways to augment PGE2‐stimulated mucociliary clearance in CF, resulting in improved clearance of pulmonary infections.Support or Funding InformationCystic Fibrosis Foundation (Z.M.S), Elizabeth Nash Foundation (BI)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.