Abstract
Abstract: :
Purpose:The hyperglycemia-induced end organ damage in diabetes is associated with production of reactive oxygen species. Ascorbic Acid plays a central role in the antioxidant defense system scavenging water-soluble reactive oxygen and nitrogen species and regenerating tocopherol from tocopheroxyl radical in membranes and lipoproteins. Ascorbic Acid uptake into the cells occurs through two major pathways, active transport by a Na+-dependent Ascorbic Acid transporters and facilitative diffusion through the glucose transporters, Glut1, 3 and 4 in oxidized Dehydroascorbic Acid form followed by immediate reduction to Ascorbic Acid. The purpose of this study was to determine the mechanism of Ascorbic Acid uptake into cells from human outer and inner blood retinal barrier; Retinal Pigment Epithelial (hRPE) and Retinal Vascular Endothelial (hRVE) cells respectively. Methods:Primary cultures of hRPE and hRVE cells were incubated in normal (5.5) and pathophysiological (22 mM) concentrations of glucose. Ascorbic Acid and Dehydroascorbic Acid uptake were measured using [14C] ascorbic acid or [14C] dehydroascorbic acid as a tracer. Dehydroascorbic acid was prepared by bromine oxidation method immediately prior to use. Glucose uptake was measured using [3H] 3-O-methylglucose as a tracer. Results:The results of our experiments demonstrate that Ascorbic Acid is transported into hRPE and hRVE cells exclusively in the Dehydroascorbic Acid form in a glucose-dependent manner. There was no Na+-dependent Ascorbic Acid uptake detected in hRPE and hRVE cells. Dehydroascorbic Acid uptake was inhibited 50% by 22 mM of glucose. Dehydroascorbic Acid competitively inhibited glucose uptake in hRPE and hRVE cells with IC50 of 1 mM. Ascorbic Acid had no effect on glucose uptake. Conclusion:hRPE and hRVE cells transport Ascorbic Acid exclusively in oxidized Dehydroascorbic Acid form in glucose-dependent manner. Thus, hyperglycemia could contribute to the development of diabetic retinopathy by depriving the retinal cells of the central antioxidant Ascorbic Acid through competition for the uptake mechanism leading to ROS accumulation. Normalizing intracellular Ascorbic Acid concentration may help to prevent ROS accumulation and consequent retinal cell damage.
Keywords: 388 diabetic retinopathy • 504 oxidation/oxidative or free radical damage • 321 antioxidants