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© 2017. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

The transmembrane receptor guanylyl cyclase‐C (GC‐C), expressed on enterocytes along the intestine, is the molecular target of the GC‐C agonist peptide linaclotide, an FDA‐approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo‐2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)‐trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase‐II (PKGII) activity assays. Expression and activity of GC‐C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo‐2BBe cells increased cell surface CFTR levels. Linaclotide‐induced activation of the GC‐C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASPser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide‐induced CFTR trafficking to the apical membrane. Inhibition of protein kinase‐A (PKA) also attenuated linaclotide‐induced CFTR cell surface trafficking, implying cGMP‐dependent cross‐activation of PKA pathway. Together, these findings support linaclotide‐induced activation of the GC‐C/cGMP/PKGII/CFTR pathway as the major pathway of linaclotide‐mediated intestinal fluid secretion, and that linaclotide‐dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.

Details

Title
Linaclotide activates guanylate cyclase‐C/cGMP/protein kinase‐II‐dependent trafficking of CFTR in the intestine
Author
Ahsan, Md Kaimul 1 ; Tchernychev, Boris 2 ; Kessler, Marco M 2 ; Solinga, Robert M 2 ; Arthur, David 3 ; Linde, Cristina I 3 ; Inmaculada Silos‐Santiago 4 ; Hannig, Gerhard 2 ; Ameen, Nadia A 5 

 Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, Connecticut 
 Department of Pharmacology, Ironwood Pharmaceuticals, Cambridge, Massachusetts 
 Biopta Ltd, Glasgow, United Kingdom 
 Department of Preclinical Research, Decibel Therapeutics, Boston, Massachusetts 
 Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, Connecticut; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 
Section
Original Research
Publication year
2017
Publication date
Jun 2017
Publisher
John Wiley & Sons, Inc.
e-ISSN
2051817X
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
1909498675
Copyright
© 2017. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.