Lubiprostone Activates Cl2 Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Mei Ao Jayashree Venkatasubramanian Chaiwat Boonkaewwan Nivetha Ganesan Asma Syed Richard V. Benya Mrinalini C. Rao
- چاپ و سال / کشور: 2010
Description
Background Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl- transport in T84 cells by activating ClC-2 channels. Aim To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl- transport. Methods Cl- transport was assessed either as Isc across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP]i was measured by enzyme immunoassay, and [Ca2?]i by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. Results Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of Isc in T84 cells. The Isc effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were*80% of those obtained with forskolin. Lubiprostone increased [cAMP]i. H89, bumetanide, or CFTRinh-172 greatly attenuated lubiprostone-stimulated Cl- secretion, whereas the ClC-2 inhibitor CdCl2 did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. Conclusions Lubiprostone activates Cl- secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.
Dig Dis Sci (2011) 56:339–351 DOI 10.1007/s10620-010-1495-8 Received: 20 July 2010 / Accepted: 10 November 2010 / Published online: 8 December 2010
