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 [¹⁴C]DHA by ARPE-19 cells was saturable with a Michaelis–Menten constant of 283µM, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid. Further, the uptakes of [¹⁴C]DHA associated with retinol-binding protein ([¹⁴C]DHA-RBP), [¹⁴C]DHA associated with low-density lipoprotein ([¹⁴C]DHA-LDL) and [¹⁴C]DHA associated with bovine serum albumin ([¹⁴C]DHA-BSA) in ARPE-19 cells increased time-dependently at 37°C, and were significantly reduced at 4°C, suggesting the involvement of energy-dependent transport processes. [¹⁴C]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 [¹⁴C]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.