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Localization of NaK ATPase on cultured human retinal pigment epithelium
1Doheny Eye Institute, Los Angeles, CA 90033.
Purpose:
To localize NaK ATPase sites on cultured human retinal pigment epithelium (RPE).
Methods:
Cultured human RPE from fetal, 2-year-old, and 21-year-old donors was grown to confluence in microporous culture wells for 4 months to 2 years, mounted in a small-volume Ussing chamber, and perfused with growth medium. Ouabain (10(-5)-M) was applied to the basal and apical sides of the RPE. Changes in transepithelial resistance (Rt), transepithelial potential (TEP), and apical and basal membrane potentials were measured.
Results:
Application of ouabain to the basal side of RPE produced a small sustained increase in TEP after 6 minutes and, simultaneously, small depolarizations of both apical and basal membranes. During the continued presence of ouabain on the basal side, application of ouabain to the apical side produced a significantly larger TEP decrease and greater depolarization of both membranes. Significant changes in Rt were not observed.
Conclusions:
These results indicate that NaK ATPase sites are present on both the apical and basolateral membranes of cultured human RPE. The greater effect of ouabain when applied to the apical side suggests that functional NaK ATPase sites are more abundant on the apical membrane.