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Chenodeoxycholate (CDCA) Activation of Ca 2+ ‐Dependent Cl ‐ Transport in Human Embryonic Kidney‐293 Cells (HEK‐293) is CFTR‐Independent
Author(s) -
George Alvin,
Domingue J,
Ao M,
Sarathy J,
Rao M.C.
Publication year - 2015
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.29.1_supplement.lb764
Subject(s) - hek 293 cells , chemistry , forskolin , cystic fibrosis transmembrane conductance regulator , receptor , endocrinology , medicine , biology , biochemistry , gene
Bile acids (BAs) are increasingly recognized to have hormone‐like functions. For example, the dihydroxy BA CDCA stimulates Cl ‐ transport in the human colonic cell line T84 via the cystic fibrosis transmembrane conductance regulator (CFTR). To explore CDCA signaling, we examined its action in CFTR‐transfected HEK (HEK‐CFTR) by iodide efflux (IE), a method used to measure Cl ‐ channel activity. CDCA (500μM)‐induced IE was not inhibited by CFTR inh 172 (20μM, n=4) in HEK‐CFTR, suggesting CDCA stimulates a non‐CFTR Cl ‐ channel in HEK. Screening for mRNA transcripts in HEK revealed expression of Ca 2+‐ activated TMEM16A, voltage‐gated ClC2, but not CFTR Cl ‐ channels ( Physiol Rep. 2(9): 1‐16, '14). Also, CDCA—but not the cAMP activator forskolin—increases IE in HEK (max IE in fold increase of baseline: CDCA: 5.8±1; Forskolin [10µM]: 3.7±0.4). To assess if CDCA action in HEK is Ca 2+ dependent (n=3), cells were pretreated with the Ca 2+ chelator BAPTA (40μM) or the IP3 receptor inhibitor 2‐APB (200μM). BAPTA and 2‐APB reduced CDCA‐induced IE by 21% and 52% respectively, indicating a role for [Ca 2+ ] i . The muscarinic M3 receptor—coupled to Ca 2+ release by G αq —has been shown to be activated by BAs and stimulate cell proliferation,but has yet to be linked to BA‐induced Cl ‐ transport. Of the muscarinic receptor isoforms (M1‐5), HEK express only M3 mRNA, but the receptor antagonist atropine (10μM) had no effect on CDCA action. Thus, CDCA‐induced IE in HEK is CFTR independent and is mediated by [Ca 2+ ] i and IP3 receptors. Our data suggest a role for TMEM16A in CDCA‐induced Cl ‐ secretion in HEK and demonstrate that HEK can serve as a model for studying BA‐action unrelated to CFTR.