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Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl− Secretion by Human Airway Epithelia

2010; Elsevier BV; Volume: 285; Issue: 45 Linguagem: Inglês

10.1074/jbc.m110.167668

1083-351X

Christian Schwarzer, Steven Wong, James Shi, Elizabeth Matthes, Beate Illek, Juan P. Ianowski, Ryan J. Arant, Ehud Y. Isacoff, Horia Vais, J. Kevin Foskett, Isabella Maiellaro, Aldebaran M. Hofer, Terry E. Machen,

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The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl− and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl− secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca2+ from the endoplasmic reticulum (ER), lowering [Ca2+] in the ER and thereby activating the Ca2+-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca2+] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl− current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca2+ buffer that lowers [Ca2+] in the ER similar to the effect of 3O-C12 also increased cAMP and ICl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl− and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca2+] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl− and fluid secretion. The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl− and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl− secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca2+ from the endoplasmic reticulum (ER), lowering [Ca2+] in the ER and thereby activating the Ca2+-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca2+] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl− current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca2+ buffer that lowers [Ca2+] in the ER similar to the effect of 3O-C12 also increased cAMP and ICl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl− and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca2+] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl− and fluid secretion. IntroductionThe Gram-negative, opportunistic bacterium Pseudomonas aeruginosa commonly infects lungs of cystic fibrosis (CF) 2The abbreviations used are: CFcystic fibrosisCFTRcystic fibrosis transmembrane regulatorERendoplasmic reticulum3O-C12N-(3-oxo-dodecanoyl)-S-homoserine lactoneTPENtetrakis-(2-pyridylmethyl)ethylenediamineIBMXisobutylmethylxanthineIP3inositol trisphosphateIP3Rinositol trisphosphate receptorCFPcyan fluorescent protein. patients and triggers innate immune responses of airway epithelial cells, including activation of NF-κB and p38 signaling and increased secretion of cytokines and chemokines that recruit white cells, primarily neutrophils, to the infected region. When CF patients become colonized with P. aeruginosa, the bacteria secrete quorum-sensing molecules, including N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) and butyryl homoserine lactone, to signal each other and to regulate their own gene expression, including genes involved in formation of biofilms (1Bjarnsholt T. Givskov M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2007; 362: 1213-1222Crossref PubMed Scopus (148) Google Scholar, 2Cooley M. Chhabra S.R. Williams P. Chem. Biol. 2008; 15: 1141-1147Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Davies D.G. Parsek M.R. Pearson J.P. Iglewski B.H. Costerton J.W. Greenberg E.P. Science. 1998; 280: 295-298Crossref PubMed Scopus (2526) Google Scholar, 4Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 5Pearson J.P. Gray K.M. Passador L. Tucker K.D. Eberhard A. Iglewski B.H. Greenberg E.P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 197-201Crossref PubMed Scopus (791) Google Scholar, 6Williams P. Microbiology. 2007; 153: 3923-3938Crossref PubMed Scopus (496) Google Scholar). Concentrations of 3O-C12 in CF sputum are thought to be in the nanomolar range (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar) but reach 5 μm in P. aeruginosa supernatants and may reach >100 μm in regions adjacent to biofilms (8Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Grauer D.C. Lehmann M. Meijler M.M. Janda K.D. Ulevitch R.J. Science. 2008; 321: 259-263Crossref PubMed Scopus (175) Google Scholar, 9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects to alter signaling and responses of multiple cell types, including human cells. 3O-C12 operates through TLR- and Nod/Ipaf/caterpillar-independent signaling to activate multiple proinflammatory genes that are associated with NF-κB signaling, including IL8, Cox2, and MUC5AC in both epithelial and other cell types (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar, 8Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Grauer D.C. Lehmann M. Meijler M.M. Janda K.D. Ulevitch R.J. Science. 2008; 321: 259-263Crossref PubMed Scopus (175) Google Scholar, 9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 10DiMango E. Zar H.J. Bryan R. Prince A. J. Clin. Invest. 1995; 96: 2204-2210Crossref PubMed Scopus (380) Google Scholar, 11Smith R.S. Fedyk E.R. Springer T.A. Mukaida N. Iglewski B.H. Phipps R.P. J. Immunol. 2001; 167: 366-374Crossref PubMed Scopus (232) Google Scholar, 12Smith R.S. Kelly R. Iglewski B.H. Phipps R.P. J. Immunol. 2002; 169: 2636-2642Crossref PubMed Scopus (131) Google Scholar). Some of these proinflammatory effects may be mediated through activation of MAPKs (8Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Grauer D.C. Lehmann M. Meijler M.M. Janda K.D. Ulevitch R.J. Science. 2008; 321: 259-263Crossref PubMed Scopus (175) Google Scholar, 9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) or Ca2+ (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar, 13Li H. Wang L. Ye L. Mao Y. Xie X. Xia C. Chen J. Lu Z. Song J. Med. Microbiol. Immunol. 2009; 198: 113-121Crossref PubMed Scopus (43) Google Scholar) or inhibition of peroxisome proliferator-activated receptor γ (14Jahoor A. Patel R. Bryan A. Do C. Krier J. Watters C. Wahli W. Li G. Williams S.C. Rumbaugh K.P. J. Bacteriol. 2008; 190: 4408-4415Crossref PubMed Scopus (116) Google Scholar). However, 3O-C12 also inhibits NF-κB signaling and expression of proinflammatory cytokines in macrophages and primary human bronchial airway epithelial cells (9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) when cells are treated with both 3O-C12 and another agonist like LPS or TNFα that activates NF-κB on its own. Thus, 3O-C12 appears to stimulate proinflammatory responses on its own but inhibit responses when present with other proinflammatory agonists.The goals of this study were to determine the effects of 3O-C12 on Cl− secretion by airway epithelia, the role for CFTR in this secretion, and whether Ca2+ and cAMP signaling were involved. 3O-C12 increases cytosolic [Ca2+] (Cacyto) in fibroblasts (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar) and mast cells (13Li H. Wang L. Ye L. Mao Y. Xie X. Xia C. Chen J. Lu Z. Song J. Med. Microbiol. Immunol. 2009; 198: 113-121Crossref PubMed Scopus (43) Google Scholar), and at least at high [3O-C12] (250–1000 μm), this resulted from Ca2+ release from an internal store, possibly the endoplasmic reticulum (ER). If 3O-C12 elicited similar effects in airway epithelia, 3O-C12 might also raise cAMP by an ER store-operated cAMP mechanism recently described for colonic epithelial cells; Lefkimmiatis (15Lefkimmiatis K. Srikanthan M. Maiellaro I. Moyer M.P. Curci S. Hofer A.M. Nat. Cell Biol. 2009; 11: 433-442Crossref PubMed Scopus (121) Google Scholar) discovered that thapsigargin (inhibitor of the Ca2+-ATPase of the ER) activated cAMP production by releasing Ca2+ from the ER, lowering [Ca2+] in the ER (CaER), and activating the ER-resident protein STIM1 (stromal interacting molecule 1; Ref 16Luik R.M. Lewis R.S. Trends Mol. Med. 2007; 13: 103-107Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and adenylate cyclase.The present experiments used electrophysiological and imaging methods to test whether the store-operated cyclase model (15Lefkimmiatis K. Srikanthan M. Maiellaro I. Moyer M.P. Curci S. Hofer A.M. Nat. Cell Biol. 2009; 11: 433-442Crossref PubMed Scopus (121) Google Scholar) could explain the stimulatory effects of 3O-C12 on Cl− secretion by airway epithelia. Transepithelial electrophysiology was used in combination with CFTR-expressing and genetically matched airway epithelial cell lines to test whether 3O-C12 increased CFTR-dependent Cl− secretion. Fluid secretion by submucosal glands in intact pig tracheas was measured to determine whether 3O-C12-stimulated Cl− secretion also contributed to fluid secretion in intact tissues. Cacyto (fura-2 imaging) and CaER (FRET imaging of ER-targeted cameleon) were measured during treatments with 3O-C12 and thapsigargin (selective blocker of Ca2+-ATPase in the ER) to test whether increases in Cacyto resulted from release of Ca2+ from the ER or from some other organelle. Patch clamp electrophysiology of inositol trisphosphate receptor 1 (IP3R1) expressed in the nuclei isolated from chicken B cells (DT40) tested whether decreases in CaER resulted from direct 3O-C12 activation of the IP3R or some other release or uptake mechanism. Total internal reflection fluorescence (TIRF) imaging was used to measure activation of STIM1, the key ER protein that has been proposed to mediate reductions in CaER to activation of cAMP production (15Lefkimmiatis K. Srikanthan M. Maiellaro I. Moyer M.P. Curci S. Hofer A.M. Nat. Cell Biol. 2009; 11: 433-442Crossref PubMed Scopus (121) Google Scholar). The role of cAMP in the Cl− secretory response was tested by measuring cAMP with Epac H30 FRET imaging and then by testing inhibitors that increase [cAMP] (phosphodiesterase blocker) and inhibit protein kinase A (Rp)-cAMP. Finally, cAMP and Cl− secretion were measured in cells treated with the membrane-permeant ER Ca2+ buffer TPEN to determine whether specific reductions in CaER (i.e. without altering Cacyto) would increase cAMP and activate Cl− secretion. IntroductionThe Gram-negative, opportunistic bacterium Pseudomonas aeruginosa commonly infects lungs of cystic fibrosis (CF) 2The abbreviations used are: CFcystic fibrosisCFTRcystic fibrosis transmembrane regulatorERendoplasmic reticulum3O-C12N-(3-oxo-dodecanoyl)-S-homoserine lactoneTPENtetrakis-(2-pyridylmethyl)ethylenediamineIBMXisobutylmethylxanthineIP3inositol trisphosphateIP3Rinositol trisphosphate receptorCFPcyan fluorescent protein. patients and triggers innate immune responses of airway epithelial cells, including activation of NF-κB and p38 signaling and increased secretion of cytokines and chemokines that recruit white cells, primarily neutrophils, to the infected region. When CF patients become colonized with P. aeruginosa, the bacteria secrete quorum-sensing molecules, including N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) and butyryl homoserine lactone, to signal each other and to regulate their own gene expression, including genes involved in formation of biofilms (1Bjarnsholt T. Givskov M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2007; 362: 1213-1222Crossref PubMed Scopus (148) Google Scholar, 2Cooley M. Chhabra S.R. Williams P. Chem. Biol. 2008; 15: 1141-1147Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Davies D.G. Parsek M.R. Pearson J.P. Iglewski B.H. Costerton J.W. Greenberg E.P. Science. 1998; 280: 295-298Crossref PubMed Scopus (2526) Google Scholar, 4Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 5Pearson J.P. Gray K.M. Passador L. Tucker K.D. Eberhard A. Iglewski B.H. Greenberg E.P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 197-201Crossref PubMed Scopus (791) Google Scholar, 6Williams P. Microbiology. 2007; 153: 3923-3938Crossref PubMed Scopus (496) Google Scholar). Concentrations of 3O-C12 in CF sputum are thought to be in the nanomolar range (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar) but reach 5 μm in P. aeruginosa supernatants and may reach >100 μm in regions adjacent to biofilms (8Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Grauer D.C. Lehmann M. Meijler M.M. Janda K.D. Ulevitch R.J. Science. 2008; 321: 259-263Crossref PubMed Scopus (175) Google Scholar, 9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects to alter signaling and responses of multiple cell types, including human cells. 3O-C12 operates through TLR- and Nod/Ipaf/caterpillar-independent signaling to activate multiple proinflammatory genes that are associated with NF-κB signaling, including IL8, Cox2, and MUC5AC in both epithelial and other cell types (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar, 8Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Grauer D.C. Lehmann M. Meijler M.M. Janda K.D. Ulevitch R.J. Science. 2008; 321: 259-263Crossref PubMed Scopus (175) Google Scholar, 9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 10DiMango E. Zar H.J. Bryan R. Prince A. J. Clin. Invest. 1995; 96: 2204-2210Crossref PubMed Scopus (380) Google Scholar, 11Smith R.S. Fedyk E.R. Springer T.A. Mukaida N. Iglewski B.H. Phipps R.P. J. Immunol. 2001; 167: 366-374Crossref PubMed Scopus (232) Google Scholar, 12Smith R.S. Kelly R. Iglewski B.H. Phipps R.P. J. Immunol. 2002; 169: 2636-2642Crossref PubMed Scopus (131) Google Scholar). Some of these proinflammatory effects may be mediated through activation of MAPKs (8Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Grauer D.C. Lehmann M. Meijler M.M. Janda K.D. Ulevitch R.J. Science. 2008; 321: 259-263Crossref PubMed Scopus (175) Google Scholar, 9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) or Ca2+ (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar, 13Li H. Wang L. Ye L. Mao Y. Xie X. Xia C. Chen J. Lu Z. Song J. Med. Microbiol. Immunol. 2009; 198: 113-121Crossref PubMed Scopus (43) Google Scholar) or inhibition of peroxisome proliferator-activated receptor γ (14Jahoor A. Patel R. Bryan A. Do C. Krier J. Watters C. Wahli W. Li G. Williams S.C. Rumbaugh K.P. J. Bacteriol. 2008; 190: 4408-4415Crossref PubMed Scopus (116) Google Scholar). However, 3O-C12 also inhibits NF-κB signaling and expression of proinflammatory cytokines in macrophages and primary human bronchial airway epithelial cells (9Kravchenko V.V. Kaufmann G.F. Mathison J.C. Scott D.A. Katz A.Z. Wood M.R. Brogan A.P. Lehmann M. Mee J.M. Iwata K. Pan Q. Fearns C. Knaus U.G. Meijler M.M. Janda K.D. Ulevitch R.J. J. Biol. Chem. 2006; 281: 28822-28830Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) when cells are treated with both 3O-C12 and another agonist like LPS or TNFα that activates NF-κB on its own. Thus, 3O-C12 appears to stimulate proinflammatory responses on its own but inhibit responses when present with other proinflammatory agonists.The goals of this study were to determine the effects of 3O-C12 on Cl− secretion by airway epithelia, the role for CFTR in this secretion, and whether Ca2+ and cAMP signaling were involved. 3O-C12 increases cytosolic [Ca2+] (Cacyto) in fibroblasts (7Shiner E.K. Terentyev D. Bryan A. Sennoune S. Martinez-Zaguilan R. Li G. Gyorke S. Williams S.C. Rumbaugh K.P. Cell. Microbiol. 2006; 8: 1601-1610Crossref PubMed Scopus (126) Google Scholar) and mast cells (13Li H. Wang L. Ye L. Mao Y. Xie X. Xia C. Chen J. Lu Z. Song J. Med. Microbiol. Immunol. 2009; 198: 113-121Crossref PubMed Scopus (43) Google Scholar), and at least at high [3O-C12] (250–1000 μm), this resulted from Ca2+ release from an internal store, possibly the endoplasmic reticulum (ER). If 3O-C12 elicited similar effects in airway epithelia, 3O-C12 might also raise cAMP by an ER store-operated cAMP mechanism recently described for colonic epithelial cells; Lefkimmiatis (15Lefkimmiatis K. Srikanthan M. Maiellaro I. Moyer M.P. Curci S. Hofer A.M. Nat. Cell Biol. 2009; 11: 433-442Crossref PubMed Scopus (121) Google Scholar) discovered that thapsigargin (inhibitor of the Ca2+-ATPase of the ER) activated cAMP production by releasing Ca2+ from the ER, lowering [Ca2+] in the ER (CaER), and activating the ER-resident protein STIM1 (stromal interacting molecule 1; Ref 16Luik R.M. Lewis R.S. Trends Mol. Med. 2007; 13: 103-107Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and adenylate cyclase.The present experiments used electrophysiological and imaging methods to test whether the store-operated cyclase model (15Lefkimmiatis K. Srikanthan M. Maiellaro I. Moyer M.P. Curci S. Hofer A.M. Nat. Cell Biol. 2009; 11: 433-442Crossref PubMed Scopus (121) Google Scholar) could explain the stimulatory effects of 3O-C12 on Cl− secretion by airway epithelia. Transepithelial electrophysiology was used in combination with CFTR-expressing and genetically matched airway epithelial cell lines to test whether 3O-C12 increased CFTR-dependent Cl− secretion. Fluid secretion by submucosal glands in intact pig tracheas was measured to determine whether 3O-C12-stimulated Cl− secretion also contributed to fluid secretion in intact tissues. Cacyto (fura-2 imaging) and CaER (FRET imaging of ER-targeted cameleon) were measured during treatments with 3O-C12 and thapsigargin (selective blocker of Ca2+-ATPase in the ER) to test whether increases in Cacyto resulted from release of Ca2+ from the ER or from some other organelle. Patch clamp electrophysiology of inositol trisphosphate receptor 1 (IP3R1) expressed in the nuclei isolated from chicken B cells (DT40) tested whether decreases in CaER resulted from direct 3O-C12 activation of the IP3R or some other release or uptake mechanism. Total internal reflection fluorescence (TIRF) imaging was used to measure activation of STIM1, the key ER protein that has been proposed to mediate reductions in CaER to activation of cAMP production (15Lefkimmiatis K. Srikanthan M. Maiellaro I. Moyer M.P. Curci S. Hofer A.M. Nat. Cell Biol. 2009; 11: 433-442Crossref PubMed Scopus (121) Google Scholar). The role of cAMP in the Cl− secretory response was tested by measuring cAMP with Epac H30 FRET imaging and then by testing inhibitors that increase [cAMP] (phosphodiesterase blocker) and inhibit protein kinase A (Rp)-cAMP. Finally, cAMP and Cl− secretion were measured in cells treated with the membrane-permeant ER Ca2+ buffer TPEN to determine whether specific reductions in CaER (i.e. without altering Cacyto) would increase cAMP and activate Cl− secretion.