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J Deguchi, A Yamamoto, T Yoshimori, K Sugasawa, Y Moriyama, M Futai, T Suzuki, K Kato, M Uyama, Y Tashiro; Acidification of phagosomes and degradation of rod outer segments in rat retinal pigment epithelium.. Invest. Ophthalmol. Vis. Sci. 1994;35(2):568-579.
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PURPOSE: The authors investigated the phagocytic processes of the rod outer segments (ROS) in rat retinal pigment epithelium (RPE) cells, and the appearance of lysosomal enzymes, acidification, and degradation of the contents in the phagolysosomes. In particular, they examined the effect of bafilomycin A1, 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 electron microscopic technique. Bafilomycin A1 was injected into the vitreous, and acidification of the phagosomes was measured in vivo by injecting 3-[2,4-dinitroanilino]3'amino-N-methyldipropylamine (DAMP) in the vitreous and detecting the accumulation of DAMP in the phagolysosomes using anti-dinitrophenol antibody. RESULTS: Opsin was abundantly detected in phagosomes that did not contain cathepsin D, but the immunolabeling 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 lysosomes. When bafilomycin A1 was injected into the vitreous, many large phagolysosomes containing cathepsin D appeared in the RPE cells, in which the immunoreactivity of opsin was well preserved. CONCLUSIONS: Degradation of opsin and acidification proceeded almost parallel with the appearance of cathepsin D in the phagolysosomes. Bafilomycin A1 did not inhibit the fusion of phagosomes with lysosomes, but it increased intraphagosomal pH and markedly inhibited the degradation of ROS in the phagolysosomes. This result indicates that vacuolar-type H(+)-ATPase is essential for acidifying the lumen of phagolysosomes and subsequent protein degradation of ROS in the RPE cells.
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