ACIDIFICATION OF PHAGOSOMES AND DEGRADATION OF ROD OUTER SEGMENTS IN RAT RETINAL-PIGMENT EPITHELIUM

Purpose. The authors investigated the phagocytic processes of the rod outer segments (ROS) in rat retinal pigment epithelium (RPE) cells, an d the appearance of lysosomal enzymes, acidification, and degradation of the contents in the phagolysosomes. In particular, they examined th e effect of bafilomycin A(1), a specific inhibitor of vacuolar-type H-ATPase, on the degradation of ROS in the RPE cells in vivo. Methods. A lysosomal enzyme (cathepsin D), a lysosomal membrane protein (LGP107 ), and opsin were localized in the RPE cells by the immunogold electro n microscopic technique. Bafilomycin A, was injected into the vitreous , and acidification of the phagosomes was measured in vivo by injectin g -[2,4-dinitroanilino]3'amino-N-methyldipropylamine (DAMP) in the vit reous and detecting the accumulation of DAMP in the phagolysosomes usi ng anti-dinitrophenol antibody. Results. Opsin was abundantly detected in phagosomes that did not contain cathepsin D, but the immunolabelin g of opsin rapidly disappeared soon after the appearance of cathepsin D. By double staining with cathepsin D and DAMP, it was shown that the pH of the phagosomes dramatically decreased after fusion with lysosom es. When bafilomycin Al was injected into the vitreous, many large pha golysosomes containing cathepsin D appeared in the RPE cells, in which the immunoreactivity of opsin was well preserved. Conclusions. Degrad ation of opsin and acidification proceeded almost parallel with the ap pearance of cathepsin D in the phagolysosomes. Bafilomycin A, did not inhibit the fusion of phagosomes with lysosomes, but it increased intr aphagosomal pH and markedly inhibited the degradation of ROS in the ph agolysosomes. This result indicates that vacuolar-type H+-ATPase is es sential for acidifying the lumen of phagolysosomes and subsequent prot ein degradation of ROS in the RPE cells.