BACKGROUND AND PURPOSE: H2 O2 is widely understood to regulate intracellular 
signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by 
activating an autocrine PGE2 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 H2 
O2 .
EXPERIMENTAL APPROACH: Anion secretion by differentiated bronchial epithelial 
cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4 
receptor agonist or β2 -adrenoceptor agonist in the presence and absence of 
inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I ) 
changes were followed by microscopy using fura-2-loaded cells and PKA activation 
followed by FRET microscopy.
KEY RESULTS: Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were 
both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial 
epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange 
protein activated by cAMP (Epac) activity that drives PLC activation to raise 
[Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to 
sAC activation and further increases in CFTR activity. Stimulation of sAC did 
not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels 
and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated 
anion currents. A similar Epac-mediated pathway was seen following β2 
-adrenoceptor or forskolin stimulation.
CONCLUSIONS AND IMPLICATIONS: H2 O2 initiated a complex signalling cascade that 
used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca(2+) 
crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a 
positive feedback loop that amplified CFTR anion secretion following stimulation 
of tmAC by a variety of stimuli.