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Identification of ANO1 (TMEM16A) activator purified from Cucumis sativus: Its role for treatment of cystic fibrosis and gastrointestinal disorders
Author(s) -
Kazi Mirajul Hoque,
Saha Tultul,
Woodward Owen M,
Guggino William B
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1224.47
Subject(s) - chloride channel , dids , chemistry , cystic fibrosis transmembrane conductance regulator , secretion , ussing chamber , xenopus , apical membrane , biochemistry , membrane , gene
BACKGROUND CFTR functions as a chloride (Cl − ) channel. Its dysfunction limits Cl − secretion leading to viscous mucus in the airway and intestinal lumen. Increases in cytosolic Ca 2+ activate the epithelial Calcium activate Chloride Channel (CaCC), which provides an alternative Cl − secretory pathway in CF. In the present study, we purified a positive fraction from Cucumis sativus and isolated compounds that activated ANO1, member of the CaCC family. METHODS The crushed fruit extracted was collected as aqueous solution by a fruit crusher followed by fractionation using High Performance Liquid Chromatography (HPLC). Short‐circuit current (Isc) and chloride (Cl − ) conductance were measured in T84 cells by Ussing chamber. ANO1 current was measured in xenopus expression system. RESULTS Administration of crude as well as purified compound to the apical side of epithelial cells activates Isc in intact as well as basolaterally permeabilized colonic T84 cells in a dose dependent manner. The stimulated Cl − secretion can be inhibited by DIDS, a calcium activated chloride channel blocker and BAPTA‐AM, an intracellular calcium channel blocker. The cAMP stimulated Cl − secretion was not altered by administration of this compound demonstrating it does not activate the CFTR mediated Cl − secretion, only the Ca 2+ activated Cl − secretion. Using a Xenopus oocyte expression system, where the oocytes express the ANO1 (TMEM16A) CaCC channel endogenously, we confirmed that both the crude and purified fractions of the C. sativus extract activate the ANO1 channel. The molecular size and fragmentation pattern of three compounds were identified by mass spectrometry. To discern the mechanism of the ANO1 activation we measured intracellular Ca 2+ levels in human bronchial epithelial cells (IB3‐1) derived from a cystic fibrosis patient, and found the active fraction of the C. sativus extract increases intracellular Ca 2+ , thereby activating ANO1 channels and Cl − secretion. Together, our data provides evidence that purified extract from C. sativus has potential therapeutic value for treatment of cystic fibrosis and gastrointestinal disorders like constipation by activating ANO1 mediated Cl − secretion in epithelial cells. Support or Funding Information Indian Council of Medical Research and Dept. of Biotechnology, Govt. of India