CFTR-Independent Prostaglandin E2-Stimulated Chloride Secretion in Human Airway.

Background: Prostaglandin E₂ (PGE₂) is a potent stimulator of airway epithelial Cl^- secretion. PGE₂ can stimulate CFTR-independent Cl^- secretion from Calu-3 submucosal gland cells, whereas human bronchial epithelial (HBE) cells require CFTR. Aim: Determine the mechanism(s) driving CFTR-independent PGE₂-stimulated Cl^- secretion in Calu-3 cells. Methods: Short-circuit current (I_sc) was measured in Calu-3, HBE, and duodenal enteroids in Ussing Chambers. mRNA expression and intracellular Ca²⁺ (Ca²⁺_i) was determined by qPCR and Fura-Red imaging, respectively. Results: In Calu-3 and HBE cells, PGE₂-stimulated I_sc was reduced by bilateral and basolateral-only removal of extracellular Ca²⁺ (Ca²⁺_e), but not by inhibition of protein kinase A (PKA), inositol 1,4,5-triphosphate (IP₃), or Ca²⁺_i stores. Duodenal enteroids utilized PKA, IP₃, Ca²⁺_i and Ca²⁺_e. EP receptor mRNA expression and functional measurements indicated EP4 receptor dominance in Calu-3 cells. EP4 receptor agonist CAY-10598 (CFTR_inh-172, glibenclamide) increased Ca²⁺_i and I_sc was driven by Ca²⁺-activated Cl^- secretion. I_sc was inhibited by dasatinib, wortmannin, and GSK650394, indicating involvement of Src, phosphoinositol phosphate (PI3K), serum glucocorticoid kinase 1 (SGK1). CFTR-independent CAY-10598-stimulated I_sc was mediated by apical Ca²⁺ release-activated Ca²⁺ channels (CRAC), P2X receptors, and basolateral TRPV channels. Conclusions: Calu-3 and HBE cells predominantly utilize EP4 receptors and Ca²⁺_e-mediated signaling for PGE₂-stimulated Cl^- secretion. However, Calu-3 cells leverage apical Ca²⁺ entry through CRAC and P2X receptors, together with basolateral TRPV activation, Src, PI3K, and SGK1 signaling, for CFTR-independent Cl^- secretion. Gaining insights into means to increase CFTR-independent airway Cl^- secretion may identify novel therapies to help ameliorate lung diseases with compromised CFTR function.