Active transport of ascorbic acid across the retinal pigment epithelium of the bullfrog

Abstract

Known functions of the RPE include glucose, water and retinoid transports; an ion transport mechanism utilizing a Na⁺- K⁺-ATPase pump located in the apical membrane has been proposed. Recent studies with cultured RPE cells of cat and bovine indicate that the RPE takes up ascorbate by an active mechanism, in this study we use a mounted bullfrog RPE preparation to study unidirectional and net fluxes of radiolabeled (¹⁴C)-ascorbic acid (AA), (¹⁴C)-dehydroascorbic acid, (³H)-L- glucose(L-glu) and (¹⁴C)-3-O-methyl-D-glucose(mD-glu) in an effort to explore the mechanism whereby AA moves across this tissue.

Comparative flux studies with AA indicated that the retina to blood side (apical to basal :AB) flux of AA was more than 6x that of L-glu, a passive marker of comparable size. The reverse BA flux of AA was not significantly different from that of L-glu. Flux studies of L-glu, mD-glu and dehydroascorbic acid revealed no “net” flux across the mounted RPE; significantly, only AA demonstrated a net flux from retina to choroid (AB). The AB flux of reduced ascorbate was significantly greater than that of dehydroascorbic acid indicating specificity of carrier mediation. Apical ouabain (10-⁻⁴ M) and sodium replacement in the bathing medium reduced the AB and net flux of AA significantly suggesting the requirement of a functioning Na⁺-K⁺-ATPase on the apical side membrane of the RPE. Energy blocker, dinitrophenol decreased unidirectional AB and net AA fluxes. Phloridzin (10-⁻⁴) administered only via the apical side of the RPE decreased net AA flux whereas phloretin and cytochalasin B, facilitated glucose carrier blockers, had no effect.

These results suggest the presence of a Na⁺/ascorbate co-transport carrier in the apical membrane of the RPE that uses the sodium gradient to move AA across the RPE from retina to blood side. It remains unclear whether in vivo the RPE moves AA to establish a gradient from the anterior to posterior regions of the eye or for some other unknown metabolic purpose of its own.