CFTR-independent prostaglandin E₂-stimulated chloride secretion in human airway

Prostaglandin E₂ (PGE₂) is a potent stimulator of airway epithelial Cl^- secretion. PGE₂ can stimulate cystic fibrosis transmembrane conductance regulator (CFTR)-independent Cl^- secretion from Calu-3 submucosal gland cells, whereas human bronchial epithelial (HBE) cells require CFTR. The aim of this study is to determine the mechanism(s) driving CFTR-independent PGE₂-stimulated Cl^- secretion in Calu-3 cells. 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. 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 used 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), and serum glucocorticoid kinase 1 (SGK1). CFTR-independent CAY-10598-stimulated I_sc was mediated by apical Ca²⁺ release-activated Ca²⁺ channels (CRACs), P2X receptors, and basolateral TRPV channels. Calu-3 and HBE cells predominantly use 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.NEW & NOTEWORTHY Identified that prostaglandin E₂ uses EP4 prostanoid receptor-mediated activation of Src, phosphoinositol phosphate (PI3K), and serum glucocorticoid kinase 1 (SGK1) to stimulate cystic fibrosis transmembrane conductance regulator (CFTR)-independent, calcium-activated chloride secretion through apical calcium release-activated calcium channels, P2X receptors, and basolateral TRPV channels in Calu-3 submucosal gland airway cells. These findings provide new potential targets to bypass airway chloride secretory defects in lung diseases with compromised CFTR function.