OR25-3 NOREPINEPHRINE INDUCES GHRELIN SECRETION BY ACTIVATION OF EPAC AND ELEVATION OF CYTOSOLIC CALCIUM CONCENTRATIONS

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR25-Signaling Originating from Membrane Receptors
Basic/Translational
Sunday, June 16, 2013: 11:15 AM-12:45 PM
Presentation Start Time: 11:45 AM
Room 133 (Moscone Center)
Bharath K Mani*1, Jen-Chieh Chuang1, Lilja Kjalarsdottir1, Joyce J Repa2 and Jeffrey Marc Zigman3
1Univerisity of Texas Southwestern Medical Center, Dallas, TX, 2University of Texas Southwestern Medical Center, Dallas, TX, 3UT Southwestern Medical Center, Dallas, TX
Ghrelin, is an orexigenic peptide secreted principally from a distinct population of gastric endocrine cells.  Molecular mechanisms regulating ghrelin secretion at the level of the ghrelin cell are mostly unknown.  Recently, norepinephrine (NE) has been shown to interact directly with β1-adrenergic receptors (β1-AR) located on ghrelin cells to enhance ghrelin release.  β1-AR is a Gs-coupled seven transmembrane receptor which elevates intracellular cAMP levels upon stimulation.  Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of calcium (Ca2+) and cAMP.  We first assessed the relative contribution of Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) and Ca2+ release from intracellular stores in regulating basal and NE-stimulated ghrelin secretion. Nifedipine, a selective L-type VGCC blocker, suppressed basal ghrelin secretion by almost 5-fold.  Quantitative PCR revealed expression of many different VGCCs, with the L-type being predominant, supporting the effect of nifedipine on ghrelin secretion.  NE-stimulated ghrelin secretion was attenuated by nifedipine only at lower NE doses (< 1 µM) but not at higher doses (10 µM).  Measurement of Ca2+ levels using fura-2 Ca2+ indicator dye suggested that NE induces release of Ca2+ from intracellular stores, as application of NE elevated [Ca2+]C in both Ca2+-containing and Ca2+-free medium. Regarding the role of cAMP, application of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor that results in accumulation of cAMP, stimulated ghrelin secretion.  Neither basal nor NE-induced ghrelin secretion was blocked by PKI, a competitive inhibitor of protein kinase A (PKA), which is a known target of cAMP.   However, pharmacologic manipulation of exchange protein activated by cAMP (EPAC), another target of cAMP, did affect ghrelin secretion.  In particular, both basal and NE-induced ghrelin secretion were attenuated by the selective EPAC inhibitor, Brefeldin A, while the EPAC agonist 8CPT-2Me-cAMP enhanced basal ghrelin secretion. We conclude that basal ghrelin secretion is primarily regulated by influx of Ca2+ across the plasma membrane via VGCCs. NE stimulates ghrelin secretion predominantly through release of intracellular Ca2+. Furthermore, cAMP and the resulting activation of EPAC are required for the normal ghrelin secretory response to NE.

Nothing to Disclose: BKM, JCC, LK, JJR, JMZ

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: An International Research alliance with The Novo Nordisk Foundation Center for Basic Metabolic Research at the University of Copenhagen (to JMZ)