title: "Store-operated" cAMP signaling contributes to Ca2+-activated Cl- secretion in T84 colonic cells.
abstract: 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 Ca(2+)-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca(2+), and the existence of apical Ca(2+)-dependent Cl(-) channels in the native colonic epithelium is controversial, leaving the identity of the Ca(2+)-activated component unresolved. We recently showed that decreasing free Ca(2+) concentration ([Ca(2+)]) 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 Ca(2+) sensor STIM1 and does not depend on changes in cytosolic Ca(2+). Here we assessed the degree to which SOcAMPS participates in Ca(2+)-activated Cl(-) transport as measured by transepithelial short-circuit current (Isc) 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 Ca(2+)-free conditions, the Ca(2+)-releasing agonist carbachol and Ca(2+) ionophore increased Isc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca(2+) chelator BAPTA-AM. The effect on Isc 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 Ba(2+) or flufenamic acid. We propose that a discrete component of the "Ca(2+)-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 Ca(2+) homeostasis, leading to diarrhea.
authors: Department of Veterans Affairs Boston Healthcare System and Department of Surgery, Brigham & Women's Hospital and Harvard Medical School, West Roxbury, Massachusetts.
authors: Jonathan M
authors: JM
authors: Nichols
authors: Department of Veterans Affairs Boston Healthcare System and Department of Surgery, Brigham & Women's Hospital and Harvard Medical School, West Roxbury, Massachusetts.
authors: Isabella
authors: I
authors: Maiellaro
authors: Department of Veterans Affairs Boston Healthcare System and Department of Surgery, Brigham & Women's Hospital and Harvard Medical School, West Roxbury, Massachusetts.
authors: Joanne
authors: J
authors: Abi-Jaoude
authors: Department of Veterans Affairs Boston Healthcare System and Department of Surgery, Brigham & Women's Hospital and Harvard Medical School, West Roxbury, Massachusetts.
authors: Silvana
authors: S
authors: Curci
authors: Department of Veterans Affairs Boston Healthcare System and Department of Surgery, Brigham & Women's Hospital and Harvard Medical School, West Roxbury, Massachusetts ahofer@rics.bwh.harvard.edu.
authors: Aldebaran M
authors: AM
authors: Hofer
mesh_terms: D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002712:Chlorides / Q000378:metabolism*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015398:Signal Transduction
chemical_list: D002712:Chlorides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D000242:Cyclic AMP; D002118:Calcium
keywords: FRET imaging; anion secretion; cAMP; colonic epithelium; endoplasmic reticulum