Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated, soluble AC-dependent cAMP amplification pathway common to GPCR signalling

Background and PurposeH(2)O(2) is widely understood to regulate intracellular signalling. In airway epithelia, H2O2 stimulates anion secretion primarily by activating an autocrine PGE(2) signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl- secretion. This study investigated signalling downstream of the receptors activated by H2O2. Experimental ApproachAnion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2O2, an EP4 receptor agonist or (2)-adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca2+](I)) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy. Key ResultsTransmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca2+](i) via Ca2+ store release (and not entry). Increased [Ca2+](i) led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3-]. Ca2+-activated apical K(Ca)1.1 channels and cAMP-activated basolateral K(V)7.1 channels contributed to H2O2-stimulated anion currents. A similar Epac-mediated pathway was seen following (2)-adrenoceptor or forskolin stimulation. Conclusions and ImplicationsH(2)O(2) initiated a complex signalling cascade that used direct stimulation of tmACs by Gs followed by Epac-mediated Ca2+ crosstalk to activate sAC. The Epac-mediated Ca2+ signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.