Fluorescent labelling of Na+, K(+)-ATPase in intact cells by use of a fluorescent derivative of ouabain: salinity and teleost chloride cells.
Anthroylouabain, a fluorescent derivative of ouabain, was used to localize Na+, K(+)-ATPase in transport epithelia of two species of teleosts. Exposure of the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) and the jaw skin of the long-jawed mudsucker (Gillichthys mirabilis) to a 2 microM anthroylouabain solution resulted in the appearance of cells stained bright blue. These were deemed to be chloride cells by their large size, distinct morphology and co-localization of DASPEI fluorescence, a mitochondrial stain. Addition of ouabain (1 mM final concentration) greatly decreased anthroylouabain fluorescent staining of chloride cells of seawater-adapted fish. Exposure of opercular membranes from freshwater tilapia to 2 microM anthroylouabain did not result in significant staining. Anthroylouabain is therefore a useful vital stain for localizing Na+,K(+)-ATPase in chloride cells of seawater-adapted teleosts, and may be useful for fluorescent labelling of ouabain-sensitive Na+,K(+)-ATPase in other tissues and species.
McCormick
S D
SD
Department of Integrative Biology, University of California, Berkeley 94720.
D000818:Animals; D000873:Anthracenes; D002712:Chlorides / Q000502:physiology*; D005399:Fishes / Q000378:metabolism*; D005456:Fluorescent Dyes; D010042:Ouabain / Q000031:analogs & derivatives*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D000873:Anthracenes; D002712:Chlorides; D005456:Fluorescent Dyes; D010042:Ouabain; C013814:anthroylouabain; D000254:Sodium-Potassium-Exchanging ATPase