Outer segment membrane turnover is difficult to study because of the small number of photoreceptors which detach their outer segments at any one time and the rapidity with which membranes are ingested. We have found that the excitatory amino acid 1- glutamate can induce massive shedding of the outer segments in the African clawed frog, the rabbit and the cat; however, the effect of this amino acid has not been well characterized. My proposal is the evaluation of 1-glutamates effects on outer segment shedding. The project will involve the induction of shedding by intra-ocular injection into mammals which possess a duplex retina and a comparison of cone and rod shedding. In parallel with these experiments, I will study the cellular mechanism of RPE ingestion with the aim of understanding how the distal outer segment membranes are removed. The large shedding response observed in the African clawed frog, rabbit, and cat provides a means of capturing all stages of outer segment detachment. In these experiments I will evaluate the role of actin in detaching the distal membranes, the formation of shielded extracellular domains as the RPE cell ingests the photoreceptor membranes, and finally, the appearance of acid phosphatase activity in the phagosome. Special emphasis will be given to the possibility that RPE cells may initiate shedding by secreting hydrolytic enzymes into the subretinal space. These experiments will be coordinated with studies on determining the role the photoreceptor plays in detaching its own distal membranes. Darkfield imaging of living photo-receptors will be used to determine if disc membrane integrity is compromised under conditions of massive shedding. In this manner, we will determine if there are structurally weak regions in the outer segment that serve as potential sites for detachment.
