title: Lubiprostone activates Cl- secretion via cAMP signaling and increases membrane CFTR in the human colon carcinoma cell line, T84.
abstract: 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.
  
  
  OBJECTIVE
  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 I(sc) 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 [Ca(2+)](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 I(sc) in T84 cells. The I(sc) 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 CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) 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.
authors.affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Mei
authors.initials: M
authors.lastname: Ao
authors.forename: Jayashree
authors.initials: J
authors.lastname: Venkatasubramanian
authors.forename: Chaiwat
authors.initials: C
authors.lastname: Boonkaewwan
authors.forename: Nivetha
authors.initials: N
authors.lastname: Ganesan
authors.forename: Asma
authors.initials: A
authors.lastname: Syed
authors.forename: Richard V
authors.initials: RV
authors.lastname: Benya
authors.forename: Mrinalini C
authors.initials: MC
authors.lastname: Rao
mesh_terms: D000527:Alprostadil / Q000031:analogs & derivatives* / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002277:Carcinoma / Q000378:metabolism*; D002400:Cathartics / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000068238:Lubiprostone; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: C482900:3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone; D001565:Benzoates; C505032:CFTR protein, human; D002400:Cathartics; D002712:Chlorides; D053778:Thiazolidines; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000068238:Lubiprostone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000527:Alprostadil