VITAMIN-A INCORPORATION INTO LIPOFUSCIN-LIKE INCLUSIONS IN THE RETINAL-PIGMENT EPITHELIUM

Intravitreal injection of the protease inhibitor leupeptin causes a ra pid accumulation of lipofuscin-like autofluorescent inclusions in the retinal pigment epithelium (RPE) of the eye. In vitamin A-deprived ani mals, similar inclusions form in response to leupeptin treatment, but they do not become autofluorescent. Because vitamin A is necessary to the development of fluorescence, it appears likely that retinoids are directly incorporated into the inclusions. Experiments were conducted to determine whether this is the case. Rats were reared on a diet cont aining retinoic acid as the only retinoid. Retinoic acid cannot be uti lized in visual transduction by the retina. When the eyes had been ove r 90%, depleted of visual cycle retinoids, the animals were given a si ngle intramuscular injection of H-3-all-trans retinol. After 7 days, w hen visual cycle retinoids had returned to an average of almost 70% of normal, the animals were given an intravitreal injection of leupeptin in each eye. At either 1 day or 7 days after the leupeptin treatment, some of the animals were dark-adapted for at least 12 h. The eyes wer e enucleated and fixed under dim red light. A region of each retina ju st superior to the optic nerve head was examined with electron microsc opic autoradiography, At both one day and 7 days after the leupeptin t reatment, the radiolabel in the RPE was primarily associated with the leupeptin induced inclusion bodies. Label was also present in the phot oreceptor outer segments. The localization of vitamin A to the leupept in-induced inclusions in the RPE strongly suggests that vitamin A is c ovalently bound to outer segment proteins that have been phagocytosed by the RPE but remain undegraded due to protease inhibition. This boun d vitamin A is probably responsible for the autofluorescence of the le upeptin-induced inclusions. Vitamin A is not likely to be bound throug h a Schiff base linkage, since retinal-Schiff base compounds do not ex hibit lipofuscin-like fluorescence.