Separation of the Rabbit Ciliary Body Epithelial Layers in Viable Form: Identification of Differences in Bicarbonate Transport

Abstract

Sections of whole ciliary (CB) dissected from Dutch belted rabbits were incubated for 2 hr at 36°C in a 30μM Ca 2+ Ringer's. This incubation resulted in the spontaneous dissociation of the two cell layers comprising this epithelium, each remaining firmly cohesive with its own basement membrane. The inner limiting membrane with its adherent non-pigmented epithelium (NPE) was then mechanically removed from the surface exposing the apical surface of the pigmented epithelium (PE). Ultrastructural examination revealed no noxious effects in most cells although gross morphological changes in the NPE cells were noted. The newly separated layers were loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. Most cells of both layers acquired stable BCECF fluorescence indicating viability. To achieve a preliminary evaluation of differences in PE and NPE bicarbonate transport, dye-loaded tissues were perfused in a flow-through chamber which was mounted on a microscope equipped for quantitative epifluorescence. The intracellular pH (pH i) of groups of cells (5-10) was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm. In Hepes-buffered Ringer's the pH is for the PE and NPE were 7·20±0·10 and 7·33±0·14 (±S.D., n = 6), respectively. Replacement of 28 mM Hepes by 28 mM HCO 3- /5% CO 2 led to a 0·13 pH i increase in the PE and a decrease of 0·27 U in the NPE. The pH i responses of the two cell layers to removal and/or reintroduction of Na + and/or Cl - were also highly dissimilar. These observations suggest that each cell type is endowed with distinct mechanisms of bicarbonate transport.