title: Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.
abstract: Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.
authors.affiliation: Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: Wersto
authors.forename: E R
authors.initials: ER
authors.lastname: Rosenthal
authors.forename: R G
authors.initials: RG
authors.lastname: Crystal
authors.forename: K R
authors.initials: KR
authors.lastname: Spring
mesh_terms: D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000256:Adenoviridae; D000818:Animals; D001692:Biological Transport; D001980:Bronchi; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000378:metabolism; D004705:Endocytosis; D004848:Epithelium; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012190:Retroviridae; D012235:Rhodamines / Q000493:pharmacokinetics; D013194:Staining and Labeling; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C505032:CFTR protein, human; D002712:Chlorides; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012235:Rhodamines; D013873:Thionucleotides; C098097:dihydrorhodamine 6G; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP