Store-operated cAMP signaling contributes to Ca2+-activated Cl- secretion in T84 colonic cells

Apical cAMP-dependent CFTR Cl- channels are essential for efficient vectorial movement of ions and fluid into the lumen of the colon. It is well known that Ca2+-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca2+, and the existence of apical Ca2+-dependent Cl- channels in the native colonic epithelium is controversial, leaving the identity of the Ca2+-activated component unresolved. We recently showed that decreasing free Ca2+ concentration ([Ca2+ ]) within the endoplasmic reticulum (ER) lumen elicits a rise in intracellular cAMP. This process, which we termed store-operated cAMP signaling (SOcAMPS), requires the luminal ER Ca2+ sensor STIM1 and does not depend on changes in cytosolic Ca2+. Here we assessed the degree to which SOcAMPS participates in Ca2+ -activated Cl- transport as measured by transepithelial short-circuit current (I-sc) in polarized T84 monolayers in parallel with imaging of cAMP and PKA activity using fluorescence resonance energy transfer (FRET)-based reporters in single cells. In Ca2+ -free conditions, the Ca2+ -releasing agonist carbachol and Ca2+ ionophore increased I-sc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca2+ chelator BAPTA-AM. The effect on I-sc was enhanced in the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), inhibited by the CFTR inhibitor CFTRinh-172 and the PKA inhibitor H-89, and unaffected by Ba2+ or flufenamic acid. We propose that a discrete component of the Ca2+ -dependent secretory activity in the colon derives from cAMP generated through SOcAMPS. This alternative mode of cAMP production could contribute to the actions of diverse xenobiotic agents that disrupt ER Ca2+ homeostasis, leading to diarrhea.