HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying bile acid (BA) regulation of Cl‐ transport. (LB720)

While human colonic T84 cells are a good model for studying Cl‐ secretion via CFTR, they respond poorly to genetic manipulations. As an alternate model, we stably transfected human embryonic kidney 293 cells (HEK), with CFTR‐EGFP (HEKCFTR) and assessed function by the I‐ efflux assay (nmol ± SEM). Forskolin (FSK; 2‐50µM) increased I‐ efflux compared to DMSO in HEKCFTR (n=3); CFTRinh‐172 (20µM) reduced FSK’s (10µM) effect by 45% (n=4). However, FSK (10µM) did not alter I‐ efflux in HEK (n=4). Disrupting microtubules with nocodazole (NOC, 33µM) did not alter FSK action in HEKCFTR (FSK: 876±103; FSK+NOC: 872±94; n=5). We had shown that the BA, chenodeoxycholic acid stimulates I‐ efflux in HEKCFTR (EB 2013). Expression studies revealed that HEK cells possess transcript and protein of the Gαs‐coupled BA receptor, TGR5. We next examined the effects of the potent TGR5 ligand, lithocholic acid (LCA) in HEKCFTR. While LCA alone had no effect on [cAMP]i it potentiated FSK’s effect (fmol/well: DMSO: 110±30; FSK: 1162±57; 50μM LCA: 146±8; FSK+ LCA: 7793±728; n=3). The functional effects of LCA on I‐ efflux were measured. Low, [10‐100μM] but not high [250‐500μM] doses of LCA increased I‐ efflux (35%, n蠅3) and pretreatment with LCA (15min, 50μM) blocked FSK’s effect (FSK: 724±66; FSK+LCA: 354±36; n=4). In summary, HEKCFTR are a good model to study CFTR because CFTR‐EGFP is functional, FSK induced efflux is CFTR specific, CFTR’s activation by FSK is not dependent on MTs and CFTR is regulated by BAs. HEKCFTR can be used to elucidate the cross talk by signals such as BAs and FSK in CFTR regulation. Grant Funding Source : NIH and VA