Abstract
Although lung disease is the major cause of mortality in cystic fibrosis (CF), gastrointestinal (GI) manifestations are the first hallmarks in 15–20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5) inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg) used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF pharmacotherapy.
Highlights
Cystic fibrosis (CF) is a life-shortening genetically inherited disease caused by mutations that alter the expression and/or the activity of the CF Transmembrane conductance Regulator (CFTR) protein
Before testing whether vardenafil can rescue CFTR-mediated chloride transport across the GI epithelia, we first determined in vivo ion transport properties of the rectal mucosa in CF mice homozygous for the F508del mutation built in the 129/FVB background [36] and in their normal homozygous littermates
Chloride transport was evaluated by the difference between the potential difference (PD) value measured at the end of perfusion with zero-chloride solution containing forskolin and that measured at the end of perfusion with Ringer solution containing amiloride and barium; it was reduced by half in F508del-CF mice (24.260.5 mV) compared to that measured in wild-type mice (29.460.9 mV; p = 0.002), integrity of chloride transport being characterized by a more marked repolarization, i.e. more negative PD values
Summary
Cystic fibrosis (CF) is a life-shortening genetically inherited disease caused by mutations that alter the expression and/or the activity of the CF Transmembrane conductance Regulator (CFTR) protein. The most prevalent F508del-CFTR mutation, present in ,70% of CF chromosomes, and in ,90% on at least one allele, of CF patients [5], corresponds to deletion of the phenylalanine 508 in the 1480 polypeptide chain It causes defective folding of the protein that is mostly retained in the endoplasmic reticulum (ER), is tagged for premature degradation by the ubiquitin-proteasomal pathway and is only marginally expressed at the surface of epithelial cells [6]. Pharmacological chaperones interacting with F508del-CFTR itself, facilitating its folding and cellular processes and agents regulating proteostasis by modulating the cellular quality-control machinery or inhibiting proteasome activity may have therapeutic potential for CF. Targeting the multiple molecular defects caused by the F508del mutation may require a therapy combining correctors and potentiators or the use of a single therapeutic agent with both correcting and potentiating properties [19,20,21]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
