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Akanuma - June 2009 Notes Biol Pharm Bull 32(6...

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Vitamin E is one of the essential micronutrients composed of lipid-soluble tocopherols and tocotrienols which play a key role in protecting the body from reactive oxygen species. 1,2) The retina is an ideal environment for the genera- tion of reactive oxygen species because light is always fo- cused on it. 3) Hence, an efficient vitamin E supply to the retina is important for protecting it from oxidative damage. a -Tocopherol is the most abundant vitamin E in the mam- malian body and it has the highest biological activity. 1) In ad- dition, a -tocopherol in plasma is associated with lipoproteins because it is hydrophobic. 4) Therefore, it is important to identify the transport mechanism of lipoprotein-associated a -tocopherol for efficient a -tocopherol supply to the retina. The nonspecific passage of substances to the retina from the circulating blood is restricted by the blood-retinal barrier (BRB), which consists of retinal pigment epithelial cells (RPE, outer BRB) and retinal capillary endothelial cells (inner BRB). 5) In contrast, the BRB expresses various trans- porters which play essential roles in supplying nutrients to the retina. 6—9) Such cumulative evidence suggests that the BRB acts as an active pathway for supplying nutrients to the retina. We recently reported that the transport of high-density lipoprotein (HDL)-associated a -tocopherol ( a -tocopherol- HDL) is mediated by scavenger receptor class B, type I (SR- BI) at the inner BRB. 10) In addition, a -tocopherol distribu- tion of RPE cells is highest in the ocular tissues of the ani- mals receiving a vitamin E-containing diet. 11) Therefore, we hypothesized that the outer BRB has an a -tocopherol-HDL transport mechanism in addition to the inner BRB. The purpose of this study was to determine the a -toco- pherol-HDL transport and clarify the contribution of SR-BI to the uptake in a cultured human RPE cell line (ARPE-19 cells). MATERIALS AND METHODS Reagents a -Tocopherol [6,7- 3 H] ([ 3 H] a -tocopherol, 60 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). [ 3 H] a -Tocopherol-HDL was generated in all experiments by incubation of an etha- nolic a -tocopherol solution with HDL from human plasma (1.5 mg protein; Merck, Darmstadt, Germany) in 2 ml extra- cellular fluid (ECF) buffer (122 m M NaCl, 25 m M NaHCO 3 , 3 m M KCl, 1.4 m M CaCl 2 , 1.2 m M MgSO 4 , 0.4 m M K 2 HPO 4 , 10 m M N -(2-hydroxyethyl)piperazine- N -2-ethanesulfonic acid (HEPES), pH 7.4) at 37 °C for 3 h. Non [ 3 H] a -toco- pherol-HDL was removed by size-exclusion chromatography on a PD-10 column (GE Healthcare, Little Chalfont, U.K.). All other chemicals used were commercially available and of reagent grade. Cell Culture ARPE-19 (American Type Culture Collec- tion, Manassas, VA, U.S.A.), the human retinal pigment ep- ithelial cell line, 12) was used to characterize the a -toco- pherol-HDL transport at the outer BRB. The ARPE-19 cell line was derived from the globes of a 19-year-old male donor. ARPE-19 cells express the mRNAs of typical RPE markers such as cellular retinaldehyde-binding protein and RPE-specific protein 65 kDa.
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