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Digestive Diseases and Sciences

, Volume 56, Issue 2, pp 339–351 | Cite as

Lubiprostone Activates Cl Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84

  • Mei Ao
  • Jayashree Venkatasubramanian
  • Chaiwat Boonkaewwan
  • Nivetha Ganesan
  • Asma Syed
  • Richard V. Benya
  • Mrinalini C. RaoEmail author
Original Article

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.

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.

Keywords

Lubiprostone Cystic fibrosis transmembrane conductance regulator cAMP signaling Cl secretion T84 

Notes

Acknowledgments

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).

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Mei Ao
    • 1
  • Jayashree Venkatasubramanian
    • 1
  • Chaiwat Boonkaewwan
    • 1
    • 3
  • Nivetha Ganesan
    • 1
  • Asma Syed
    • 1
  • Richard V. Benya
    • 2
  • Mrinalini C. Rao
    • 1
    Email author
  1. 1.Department of Physiology and BiophysicsUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of MedicineUniversity of Illinois at ChicagoChicagoUSA
  3. 3.Department of Animal ScienceKasetsart UniversityBangkokThailand

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