PURPOSE: Alpha-Tocopherol is an essential micronutrient acting as an antioxidant in the retina. However, the molecular mechanism of its retinal uptake from the circulating blood remains to be determined. The purpose of this study was to elucidate the contribution of scavenger receptor class B, type I (SR-BI), to the uptake of high-density lipoprotein (HDL)-associated alpha-tocopherol (alpha-tocopherol-HDL) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner blood-retinal barrier model. METHODS: An uptake study of alpha-tocopherol-HDL was performed using TR-iBRB2 cells. The expression of SR-BI protein was determined by immunoblot and immunohistochemical analyses. RNA interference was done to clarify the relationship between SR-BI protein expression and the uptake of alpha-tocopherol-HDL by TR-iBRB2 cells. RESULTS: [(14)C]alpha-tocopherol-HDL uptake by TR-iBRB2 cells exhibited a time-dependent increase and a temperature-dependence with an 88% reduction for 90 min at 4 degrees C compared with that at 37 degrees C. The uptake of [(14)C]alpha-tocopherol-HDL was inhibited by BLT-1, a specific inhibitor of the SR-BI-mediated lipid transfer between HDL and cells, in a concentration-dependent manner with an IC(50) of 23.2 nM. SR-BI protein expression was detected in TR-iBRB2 cells and SR-BI immunostaining was observed along the rat retinal capillaries. Inhibition of SR-BI protein expression by SR-BI siRNA resulted in a 24.4% reduction in [(14)C]alpha-tocopherol-HDL uptake. CONCLUSIONS: Our findings strongly suggest that SR-BI at the inner blood-retinal barrier is responsible for alpha-tocopherol uptake from the circulating blood and plays a key role in maintaining alpha-tocopherol in the neural retina.
PURPOSE:Alpha-Tocopherol is an essential micronutrient acting as an antioxidant in the retina. However, the molecular mechanism of its retinal uptake from the circulating blood remains to be determined. The purpose of this study was to elucidate the contribution of scavenger receptor class B, type I (SR-BI), to the uptake of high-density lipoprotein (HDL)-associated alpha-tocopherol (alpha-tocopherol-HDL) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner blood-retinal barrier model. METHODS: An uptake study of alpha-tocopherol-HDL was performed using TR-iBRB2 cells. The expression of SR-BI protein was determined by immunoblot and immunohistochemical analyses. RNA interference was done to clarify the relationship between SR-BI protein expression and the uptake of alpha-tocopherol-HDL by TR-iBRB2 cells. RESULTS: [(14)C]alpha-tocopherol-HDL uptake by TR-iBRB2 cells exhibited a time-dependent increase and a temperature-dependence with an 88% reduction for 90 min at 4 degrees C compared with that at 37 degrees C. The uptake of [(14)C]alpha-tocopherol-HDL was inhibited by BLT-1, a specific inhibitor of the SR-BI-mediated lipid transfer between HDL and cells, in a concentration-dependent manner with an IC(50) of 23.2 nM. SR-BI protein expression was detected in TR-iBRB2 cells and SR-BI immunostaining was observed along the rat retinal capillaries. Inhibition of SR-BI protein expression by SR-BI siRNA resulted in a 24.4% reduction in [(14)C]alpha-tocopherol-HDL uptake. CONCLUSIONS: Our findings strongly suggest that SR-BI at the inner blood-retinal barrier is responsible for alpha-tocopherol uptake from the circulating blood and plays a key role in maintaining alpha-tocopherol in the neural retina.