Multiple Cellular Transport and Binding Processes of Unesterified Docosahexaenoic Acid in Outer Blood-Retinal Barrier Retinal Pigment Epithelial Cells

Docosahexaenoic acid (DHA, 22:6) is an essential omega-3 long-chain polyunsaturated fatty acid that plays a pivotal role in vision. The purpose of this study was to clarify the cellular uptake and binding processes of free and protein-bound unesterified DHA in retinal pigment epithelial cell (RPE) line ARPE-19 as a model of the human outer blood retinal barrier and isolated porcine RPE cell fractions. Uptake of free [C-14]DHA by ARPE-19 cells was saturable with a Michaelis Menten constant of 283 mu m, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid. Further, the uptakes of [C-14]DHA associated with retinol-binding protein ([C-14]DHA-RBP), [C-14]DHA associated with low-density lipoprotein ([C-14]DHA-LDL) and [C-14]DHA associated with bovine serum albumin ([C-14]DHA-BSA) in ARPE-19 cells increased time-dependently at 37 degrees C, and were significantly reduced at 4 degrees C, suggesting the involvement of energy-dependent transport processes. [C-14]DHA-LDL uptake by ARPE-19 cells was significantly inhibited by excess unlabeled LDL, but not by an inhibitor of scavenger receptor B type I. Fatty acid transport protein (FATP) 2 and 4 mRNAs were expressed in ARPE-19 cells, and [C-14]DHA uptake was observed in FATP2- and FATP4-expressing Xenopus oocytes. Photo-reactive crosslinking and mass spectrometry analyses identified 65-kDa retinal pigment epithelium-specific protein (RPE65) as a DHA-binding protein in porcine RPE cell membrane fractions. Thus, RPE cells possess multiple cellular transport/binding processes for unesterified DHA, involving at least partly FATP2, FATP4, LDL, RBP, and RPE65.