The chick retinal pigment epithelium grown on permeable support demonstrates functional polarity

Abstract

The retinal pigment epithelium (RPE) from the chick embryo was cultured on permeable support. Using confluent cultures and analysis of the incubation medium, the present study demonstrates that RPE cells cultured on permeable membrane retain functional polarity, a characteristic of the RPE in vivo. The degree of intercellular permeability in the confluent RPE cultures was estimated by following [ 3H]inulin movement from the apical side to the basal side of the cultures. Twenty-four hours after exposure of the apical side of the culture to [ 3H]inulin, the 3H concentration in the apical medium remained at 3.4 to 4.4 times of that in the basal medium. The barrier function of RPE disappears in the presence of EDTA. Net unidirectional fluid movement from the apical side of the cultures to the basal side of the cultures is regularly observed in confluent RPE cultures. The rate varies among different preparations of cultures and the highest is 1.60–1.84 μl/cm 2/h. When cultures are given 26 h of [ 35S]methionine, more than 20 bands with molecular weights ranging from 20,000 to >250,000 Da can be detected in the medium as assessed by autoradiography of SDS-polyacrylamide gels. While six macromolecules appear to be equally concentrated in the basal medium and the apical medium, the majority are in higher concentration in the basal medium. Analysis of the 10% TCA-precipitable fraction of the medium showed that the specific activities in the apical medium and basal medium were 24.0±0.4×10 6 and 46.4±0.2×10 6 (mean ± SEM, N = 8) cpm/ml/mg RPE protein, respectively. When cultures react with VIP (vasoactive intestinal peptide), the elevated intracellular cyclic AMP is extruded into the medium bathing the cells. However, the rate of extrusion into the basal medium is twice as fast as that into the apical medium. Electron microscopy of the confluent RPE cultures shows morphological polarization of the cells. The intercellular spaces appear to be closed at the apical side of the cells by junctional complexes consisting of tight junctions, zonular adherens junctions, and gap junctions.