caa a22 4500 445797568 CHVBK 20180317145130.0 cr unu---uuuuu 170323e20110201xx s 000 0 eng 10.1007/s10620-010-1495-8 doi (NATIONALLICENCE)springer-10.1007/s10620-010-1495-8 Lubiprostone Activates Cl− Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84 [Elektronische Daten] [Mei Ao, Jayashree Venkatasubramanian, Chaiwat Boonkaewwan, Nivetha Ganesan, Asma Syed, Richard Benya, Mrinalini Rao] 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. Springer Science+Business Media, LLC, 2010 Lubiprostone nationallicence Cystic fibrosis transmembrane conductance regulator nationallicence cAMP signaling nationallicence Cl− secretion nationallicence T84 nationallicence Ao Mei Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Venkatasubramanian Jayashree Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Boonkaewwan Chaiwat Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Ganesan Nivetha Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Syed Asma Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Benya Richard Department of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Rao Mrinalini Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut Digestive Diseases and Sciences Springer US; http://www.springer-ny.com 56/2(2011-02-01), 339-351 0163-2116 56:2<339 2011 56 10620 https://doi.org/10.1007/s10620-010-1495-8 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10620-010-1495-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ao Mei Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 700 1- Venkatasubramanian Jayashree Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 700 1- Boonkaewwan Chaiwat Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 700 1- Ganesan Nivetha Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 700 1- Syed Asma Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 700 1- Benya Richard Department of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 700 1- Rao Mrinalini Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, 60612, Chicago, IL, USA aut NATIONALLICENCE 773 0- Digestive Diseases and Sciences Springer US; http://www.springer-ny.com 56/2(2011-02-01), 339-351 0163-2116 56:2<339 2011 56 10620 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer