The proposed research focuses on the differences in fatty acid composition between the retinal pigment epithelium (RPE) and photoreceptors. These differences in amounts of saturated and unsaturated fatty acids reflect the ability of the RPE and/or retina to sort out plasma-derived fatty acids for specific destinations. This proposal addresses some of the mechanisms by which these differences are maintained. In one phase of the proposal, the relative abilities of the retina and RPE to metabolize fatty acids is investigated. In the second phase, the role of fatty acid binding protein (FABP) is examined.
These aims will be addressed by the following experiments. In the first phase, the uptake and metabolism of radiolabelled oleic, linoleic, and linolenic acid by porcine retinas and isolated RPE cells will be measured. Following the incubation in vitro, lipids will be extracted and separated by thin-layer chromatography. Fatty acid products will be separated by radio-gas-liquid chromatography. From these analyses, the extent to which these substrates and products are directed into phospholipids and other lipid classes, or if the fatty acids remain in the free fatty acid pool, can be evaluated. In addition, the rates of desaturation and chain elongation of fatty acids can be estimated and compared between retina and RPE. In the second phase, FABP will be purified from porcine retinas and the endogenously bound fatty acids will be analyzed. The binding constants for a variety of fatty acids and other hydrophobic substrates will be determined to assess the specificity and preference of this carrier protein. The effect of FABP on two retinal enzymes, lysophospholipid acyltransferase and retinyl ester synthetase, will be measured. An understanding of the way fatty acids are handled by these ocular tissues will contribute to our knowledge of the communication system between the retina and RPE, especially with respect to the transport of hydrophobic substrates. These experiments may also clarify the susceptibility of the retina to light damage by defining the step where recognition of a particular (or possibly damaged) fatty acid could take place.
