Na⁺- Cl^-- and HCO^-₃ dependent Base Uptake in the Ciliary Body Pigment Epithelium

Segments of whole ciliary body dissected from Dutch belted rabbits were incubated for 60 min at 36°C in a 30 μM Ca²⁺ Ringer's. The inner limiting membrane with its adherent non-pigmented epithelium then was mechanically removed from the surface. The newly-exposed viable layer of pigmented cells was then loaded with the fluorescent probe 2′-7′-bis (carboxymethyl)-5(6) carboxyfluorescein by incubating the segments for 45 min at RT with the cell permeable acetoxymethoxy form of the dye. These loaded tissues were perfused in a flow-through chamber, mounted on the heated stage of a microscope equipped for quantitative epifluorescence, and the intracellular pH (pH₁) of small groups of cells was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm, respectively. In N[2-hydroxyethyl] piperazine-N″-[2 ethane sulfonic acid] (Hepes)-buffered Ringer's the intracellular pH was 7·23 ± 0·21 (± S.D., n = 20). Replacement of 28 mM Hepes by 28 mM HCO^-₃/5 % CO₂ led to a 0·14 ± 0·04 increase in pH₁. This increase required the presence of Na⁺ and Cl^- and was inhibited by 0·2 mM diisothiocyanatostilbene-2-2′-disulfonic acid. These observations as well as characteristic pH₁ responses to the removal or introduction of Na⁺ or Cl^- indicated the presence in the pigmented cells of a Na⁺- and Cl-^- dependent HCO^-₃ transporter responsible for base uptake.