Apical Electrogenic NaHCO Cotransport A Mechanism for HC03 Absorption across the Retinal Pigment Epithelium

Intracellular microelectrode techniques and intraceUular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCOs]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (V~). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCOs]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco~ (and consequently [HCO3]I) by the addition of apical NH4 (20 mM) produced an immediate depolarization of V,r This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 contransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCOs]i), indicating that the transporter moves NaHCOs and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering V~ with transepithelial current and then measuring the DIDS-induced change in V~. Depolarization of V~ increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond 1 1 4 mV caused the DIDSinduced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na). I N T R O D U C T I O N The retinal pigment epithelium (RPE) is a single layer of cells that forms an important part o f the b lood-re t ina barr ier and provides the major t ransport pathway Address reprint requests to Dr. Sheldon S. Miller, 360 Minor Hall, University of California, Berkeley, CA 94720. Dr. Hughes' present address is Department of Physiology, S-762, University of California, San Francisco, CA 94143. J. GEN. PHYSIOL. ~ The Rockefeller University Press 9 0022-1295/89/07/0125/26 $2.00 Volume 94 July 1989 125-150 125 on M ay 9, 2017 D ow nladed fom Published July 1, 1989