Linaclotide activates guanylate cyclase‐C/cGMP/protein kinase‐II‐dependent trafficking of CFTR in the intestine. Issue 11 (7th June 2017)

• Record Type: Journal Article
• Authors: Ahsan, Md. Kaimul; Tchernychev, Boris; Kessler, Marco M.; Solinga, Robert M.; Arthur, David; Linde, Cristina I.; Silos‐Santiago, Inmaculada; Hannig, Gerhard; Ameen, Nadia A.
• Is Part Of: Physiological reports. Volume 5:Issue 11(2017)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-06-07
• Subjects: CFTR -- chronic idiopathic constipation -- irritable bowel syndrome with constipation -- linaclotide -- PKGII/PKA; Physiology -- Periodicals; 571
• DOI: 10.14814/phy2.13299
• Languages: English
• ISSNs: 2051-817X

Abstract

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 (PKG‐II) 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 pVASP ser239 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/PKG‐II/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. Abstract : Linaclotide has been shown to increase intestinal fluid secretion by cGMP activation of CFTR. However, whether trafficking of CFTR is involved remains unknown. This study elucidates the trafficking mechanism and confirms that linaclotide activates CFTR by stimulating its traffic into the brush border membrane.