Seonghee Park1, Nikolay Shcheynikov2, Jeong Hee Hong2, Changyu Zheng2, Suk Hyo Suh3, Katsuhiro Kawaai4, Hideaki Ando4, Akihiro Mizutani5, Takaya Abe6, Hiroshi Kiyonari6, George Seki7, David Yule8, Katsuhiko Mikoshiba4, Shmuel Muallem9. 1. Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland; Department of Physiology, School of Medicine, Ewha Womans University, Seoul, Republic of Korea. 2. Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland. 3. Department of Physiology, School of Medicine, Ewha Womans University, Seoul, Republic of Korea. 4. Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan. 5. Department of Pharmacotherapeutics, Showa Pharmaceutical University, Machida, Tokyo, Japan. 6. Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan. 7. Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. 8. Department of Pharmacology & Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York. 9. Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland. Electronic address: Shmuel.muallem@nih.gov.
Abstract
BACKGROUND & AIMS: The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. METHODS: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses. RESULTS: Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. CONCLUSIONS: Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.
BACKGROUND & AIMS: The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mousepancreatic and salivary gland ducts. METHODS: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mousepancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses. RESULTS:Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. CONCLUSIONS:Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.
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