Bioelectric Characterization of Epithelia from Neonatal CFTR Knockout Ferrets

Abstract

Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (I SC ) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,49-diisothiocyano-2,29-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene) glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible I SC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/ forskolin I SC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

Lung Phenotype of Juvenile and Adult Cystic Fibrosis Transmembrane Conductance Regulator–Knockout Ferrets

“…Unfortunately, electrophysiologic studies were not performed on the third CF animal (CF-1), which was also over 250 days old. Studies in newborn CF pig tracheas failed to demonstrate changes in ENaC activity (24), and this is similar to observations in newborn CF ferrets (25). Although the number of older animals with enhanced amiloride-sensitive tracheal currents remains low, the link between enhanced ENaC activity and progression of airway disease in CF ferrets warrants further investigation.…”

Origins of Cystic Fibrosis Lung Disease

“…Those results were expected because CFTR is an apical membrane anion channel regulated by phosphorylation with cAMP-dependent protein kinase. These findings correspond with studies in the cystic fibrosis ferret 21,32 , rat 22 , and human 2 airway epithelia, which consistently demonstrate a loss of anion permeability. In addition, the salt concentration in airway surface liquid was similar between newborn noncystic fibrosis and cystic fibrosis pigs 45 .…”

“…A similar gene targeting strategy was used to produce ferrets lacking CFTR 21 . They develop characteristic features of human cystic fibrosis including intestinal, pancreatic, and airway disease, and they may be particularly valuable for studying cystic fibrosis-related diabetes mellitus 21,[31][32][33][34] . Cystic fibrosis rats, recently produced using zinc finger endonuclease technology, develop intestinal, airway and reproductive features consistent with human disease 22 .…”

Testosterone regulates levels of cystic fibrosis transmembrane regulator, adenylate cyclase, and cAMP in the seminal vesicles of orchidectomized rats

“…Patch clamp experiment can be used to investigate this relationship [31][32][33]. In patch-clamp study, the magnitude of a short circuit current (Isc) in the cultured epithelial monolayer that can be obtain from seminal vesicles of animals receiving different treatment (as above) can be measured.…”