Premium
Prostaglandin E 2 ‐Stimulated Cl − Secretion in Airway Epithelium Involves Protein Kinase A and Calcium Crosstalk
Author(s) -
Abazari Shayda,
Hari Gopika,
Crowther Elizabeth,
Kiles Franny,
Trumbull Annie,
Illek Beate,
Sellers Zachary M.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.734.3
Subject(s) - cystic fibrosis transmembrane conductance regulator , secretion , mucociliary clearance , medicine , ussing chamber , endocrinology , cystic fibrosis , chemistry , receptor , agonist , epithelium , lipoxin , prostaglandin , lung , biology , pathology
Background Mucociliary clearance is an integral defense mechanism against pulmonary infections and is impaired in Cystic Fibrosis (CF) airways. Prostaglandin E 2 (PGE 2 ) is abundant in infected airways and stimulates transepithelial Cl − secretion and mucociliary clearance, both of which are impaired in CF. Previously, we found that PGE 2 ‐stimulated Cl − secretion was cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent in bronchial epithelial cells, but in submucosal gland cells utilized both CFTR and Ca 2+ ‐activated Cl − channels (CaCC). Aim To determine the signaling mechanism(s) whereby PGE 2 stimulates airway Cl − secretion in order to identify ways to circumvent the mucociliary defect in CF. Methods Human bronchial epithelial cell cultures (CFBE41o‐ with wildtype CFTR) were used for all experiments. Clsecretion was measured by short‐circuit current ( I sc ) 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 I sc reflects primarily Cl − transport. EP receptor expression was determined by qPCR. Results In bronchial epithelial cell cultures, qPCR showed that EP 4 receptor expression was 148% greater than EP 3 receptor expression. CAY10598 (1 μM), a selective EP 4 agonist, stimulated Cl − secretion to a similar magnitude as PGE 2 (Cay: 85.5 +/− 8.6 μA/cm 2 (n=4) vs. PGE 2 : 81.6 +/− 10.2 μA/cm 2 (n=17); mean +/− SE). In contrast, sulprostone, an EP 3 agonist, was less effective at 1 or 10 μM (5.6+/− 2.0 μA/cm 2 (n=6) and 14.5+/− 7.3 μA/cm 2 (n=2), respectively). Pre‐treatment with H‐89 (10 μM), a protein kinase A (PKA) inhibitor, decreased PGE 2 ‐stimulated Cl − secretion by 74% (n=9). Similarly, pretreatment with BAPTA‐AM (100 μM), a Ca 2+ chelator, inhibited PGE 2 ‐stimulated Cl − secretion by 71% (n=7). Simultaneous pre‐treatment with H‐89 and BAPTA‐AM was additive and completely abolished PGE 2 ‐stimulated Cl − secretion (94% inhibition, n=3). Additionally, wortmannin (1μM), a phosphoinositide 3‐kinase (PI3K) inhibitor, decreased PGE 2 ‐stimulated Clsecretion by 56% (n=4). Summary and Conclusions PGE 2 ‐stimulated Cl − secretion in human bronchial epithelial cells predominantly occurs via EP 4 receptors. While utilizing classic EP 4 receptor signaling (PKA and PI3K), there is also substantial crosstalk with intracellular Ca 2+ . Interestingly, the latter does not activate CaCC, but rather CFTR. Ongoing experiments are aimed at further delineating the intracellular signaling mechanisms involved in PGE 2 ‐stimulated Cl − secretion in bronchial epithelial cells, as well as comparing these mechanisms to PGE 2 ‐stimulated Cl − secretion in submucosal gland cells. Through this we hope to identify potential ways to augment PGE 2 ‐stimulated mucociliary clearance in CF, resulting in improved clearance of pulmonary infections. Support or Funding Information Cystic 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 .