title: Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line.
abstract: Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.
authors.affiliation: Center for Cell and Molecular Signaling, Department of Medicine, Emory University, and Department of Veterans Affairs Medical Center, Atlanta, Georgia 30322, USA.
authors.forename: B N
authors.initials: BN
authors.lastname: Ling
authors.forename: J B
authors.initials: JB
authors.lastname: Zuckerman
authors.forename: C
authors.initials: C
authors.lastname: Lin
authors.forename: B J
authors.initials: BJ
authors.lastname: Harte
authors.forename: K A
authors.initials: KA
authors.lastname: McNulty
authors.forename: P R
authors.initials: PR
authors.lastname: Smith
authors.forename: L M
authors.initials: LM
authors.lastname: Gomez
authors.forename: R T
authors.initials: RT
authors.lastname: Worrell
authors.forename: D C
authors.initials: DC
authors.lastname: Eaton
authors.forename: T R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000503:physiopathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000187:drug effects; D008954:Models, Biological; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010641:Phenotype; D012964:Sodium / Q000502:physiology; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D016376:Oligonucleotides, Antisense; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000584:Amiloride; D012964:Sodium