Purpose: To determine whether the temperature-sensitive rat retinal pigment epithelial cell line (RPE-J) express mRNA transcripts for apolipoprotein(apo) and microsomal triglyceride transfer protein (MTP) and whether it can synthesize and secrete neutral lipids.
Methods: RPE-J cells were cultured in plastic tissue culture flasks and on Transwell filters. RT-PCR was used to check mRNA expression. Phagocytosis was checked by adding FITC-labeled beads and measuring the intensity of the fluorescence. Lipids were extracted from cultured cells that were fed photoreceptor outer segments (OS) for 24h; thin layer chromatography and enzymatic fluorimetry were used to assay lipid content. Cells were supplemented with [3H]-oleate (5000 dpm/nmole) bound to 1.5% fatty acid free bovine serum albumin in order to permit detection of the new synthesis and secretion of neutral lipids.
Results: RPE-J cells express mRNA for apoA-I, apoA-II, apoB, apoC-I, II, III, and apo E, as well as MTP. RPE-J cells can phagocytose OS and beads. OS-fed RPE-J cells, relative to untreated cells, have 5.8 times more triglyceride, 2.6 times more phospholipid, 1.5 times more unesterified cholesterol, and 0.3 times more esterified cholesterol. Radiolabeled neutral lipid is detected in cells and medium. All lipids increase in cells with time but only phospholipid increases in the medium with time.
Conclusions: RPE cells express genes encoding apolipoproteins and MTP. Following the ingestion of OS, RPE-J cells contain abundant lipid. Following the ingestion of long-chain fatty acids, RPE-J cells can synthesize neutral lipids. Future experiments will determine if RPE-J cells secrete lipoprotein particles containing these proteins.