Lubiprostone Activates Cl− Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84
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.
To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl− transport.
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.
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.
Lubiprostone activates Cl− secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.
KeywordsLubiprostone Cystic fibrosis transmembrane conductance regulator cAMP signaling Cl− secretion T84
This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (1 P01 DK067887 Project 3) (to MC Rao and RV Benya), and National Institute of Health Grant DK 58135 (to MC Rao).
- 1.Perentesis G, Crawford D, Engelke K. In vivo metabolism of lubiprostone, a novel GI chloride channel activator, in mouse, rat, dog, and monkey. Gastroenterology. 2005; 100:A904.Google Scholar
- 3.Ueno R, Osama H, Habe T. Oral SPI-0211 increases intestinal fluid secretion and chloride concentration without altering serum electrolyte levels. Gastroenterology. 2004; 126:A298.Google Scholar
- 25.Britton FC, Hatton WJ, Rossow CF, Duan D, Hume JR, Horowitz B. Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues. Am J Physiol. 2000;279:H2225–H2233.Google Scholar
- 27.Lipecka J, Bali M, Thomas A, Fanen P, Edelman A, Fritsch J. Distribution of ClC-2 chloride channel in rat and human epithelial tissues. Am J Physiol. 2002;282:C805–C816.Google Scholar