Cl- permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl- permeability in cystic fibrosis.
Salt reabsorption by the human sweat duct is markedly reduced in cystic fibrosis (CF). We used fluorescence-digital imaging microscopy in combination with a halide-specific fluorescent dye [6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] to determine if this defective salt reabsorption is referable to a reduced plasma membrane Cl- permeability of the epithelial cells that line the sweat duct. Sweat duct cells were cultured from explants of normal and CF reabsorptive duct and loaded with SPQ, the fluorescence of which is specifically quenched by halide ions (Br- greater than Cl-) and provides a relative index of intracellular halide concentration. Two lines of evidence indicate that normal sweat duct cells exhibit a substantial permeability to Cl- and Br-. First, the replacement of extracellular Cl- with an impermeant anion (i.e., gluconate) resulted in a rapid and reversible increase in the intracellular fluorescence, as expected if the cells rapidly lost Cl- to the extracellular media. Second, the replacement of extracellular Cl- with Br- resulted in a rapid and reversible quenching of the intracellular fluorescence, as expected if the cells accumulated Br- (a more effective quencher of SPQ fluorescence) in exchange for Cl-. The rate of fluorescence change that was induced by either maneuver was inhibited by the Cl- channel blocker, diphenylamine-2-carboxylate (10 microM). Moreover, CF cells exhibited markedly reduced rates of fluorescence change in response to either maneuver. Our results document the utility of this imaging strategy for assessing the Cl- permeabilities of individual epithelial cells that are affected in cystic fibrosis and indicate that the defective salt reabsorption by the CF sweat duct is referable, at least in part, to a reduced plasma membrane Cl- permeability of sweat duct cells.
Ram
S J
SJ
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Kirk
K L
KL
D000293:Adolescent; D000328:Adult; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D012016:Reference Values; D013545:Sweat Glands / Q000166:cytology / Q000378:metabolism* / Q000473:pathology
D002712:Chlorides