Na(+-) Cl(-)- and HCO3(-)-dependent base uptake in the ciliary body pigment pigment epithelium

Segments of whole ciliary body dissected from Dutch belted rabbits were incubated for 60 min at 36 degrees C in a 30 microM 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 (pHi) 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 HCO3-/5% CO2 led to a 0.14 +/- 0.04 increase in pHi. 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 pHi, responses to the removal or introduction of Na+ or Cl- indicated the presence in the pigmented cells of a Na(+)- and Cl(-)-dependent HCO3- transporter responsible for base uptake.