Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.
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.
Wersto
R P
RP
Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Rosenthal
E R
ER
Crystal
R G
RG
Spring
K R
KR
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
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