Membrane Polarity of the Na+-K+ Pump in Primary Cultures of Xenopus Retinal Pigment Epithelium

Abstract

The retinal pigment epithelium is a transporting epithelium that helps regulate the volume and composition of the subretinal space surrounding photoreceptor outer segments. The capacity of the RPE to actively transport Na+ and K+ between the retina and the blood supply depends on the localization of the Na+, K+-ATPase to the apical membrane, but in culture this polar distribution can be lost. Using primary cultures of Xenopus RPE, we examined the anatomical and functional polarity of this electrogenic pump. Confluent monolayers were established on Matrigel-coated microporous filters and cultured for 2-4 weeks in serum-free defined medium. Electrogenic pump activity at the apical and basolateral membranes was assayed by mounting the monolayer and filter in an Ussing chamber and exposing one or the other surface to ouabain while recording the apical (Vap) and basolateraI (Vba) membrane potentials with an intracellular microelectrode. The addition of 0[middle dot]2 mM ouabain to the apical bath caused Vap to rapidly depolarize by about 4 mV, consistent with the inhibition of a hyperpolarizing pump current at that membrane. When ouabain was added to the basal bath, however, it had no effect on Vba, suggesting the absence of a functional Na+-K+ pump on the basolateral membrane. To confirm these electrophysiological results, we examined the distribution of the Na+, K+-ATPase catalytic component using an antiserum specific for the bovine kidney [alpha] subunit. Antibody labeling of cultures was highly polarized, with strong reaction present on the apical microvilli, but not the basolateral cell surfaces. The findings of this study indicate that the Na+-K+ pump in monolayers of Xenopus RPE, as in native RPE, is located mainly in the apical membrane, providing evidence of a functionally intact transport pathway in these primary cultures.