PURPOSE: To characterize the properties of the glucose transporters of bovine retinal capillary endothelial cells and pericytes and to determine the effects of increased glucose concentrations on glucose transport activity. METHODS: Primary cultures of bovine retinal capillary endothelial cells and pericytes were exposed to low and high glucose concentrations, and immunoblot analysis, 14C-3-O-methylglucose transport activity, and cytochalasin B binding assays were used to characterize the glucose transporters. RESULTS: GLUT1, but not GLUT3 or GLUT4 transporter isoforms, was present in plasma membranes isolated from each cell type. The EC50 for glucose transport was similar in endothelial cells and pericytes (3.94 to 0.48 mM versus 2.24 to 0.69 mM) and was consistent with the EC50 previously reported for GLUT1 transporters on other cells, as was the observation that insulin did not acutely stimulate glucose transport in either cell type. The Vmax for glucose transport was greater in pericytes than endothelial cells (71 to 25 versus 14.5 to 0.8 pmol/10 s/g DNA). Exposure of pericytes to 20 mM glucose for 8 days decreased the initial maximal rate of glucose transport by 30%, compared to pericytes cultured in 5 mM glucose (187 to 7 versus 133 to 9 fmol/20 s/g DNA, P < 0.01), but had no effect on glucose transport activity in endothelial cells. Culture in high glucose decreased the apparent amount of immunoreactive pericyte plasma membrane GLUT1 in immunoblots (0.611 to 0.055 versus 1.0 relative density units), decreased the binding of 3H-cytochalasin B to pericyte plasma membranes, and decreased the mRNA level for GLUT1 in pericytes by 25%. CONCLUSIONS: High-glucose concentrations downregulate glucose transport activity and GLUT 1 content in retinal capillary pericytes but not in endothelial cells. This effect occurred at a pretranslational level. The selective effects of high-glucose concentrations on retinal capillary pericytes in culture might be related to the selective effects of hyperglycemia on these cells in vivo.
PURPOSE: To characterize the properties of the glucose transporters of bovine retinal capillary endothelial cells and pericytes and to determine the effects of increased glucose concentrations on glucose transport activity. METHODS: Primary cultures of bovine retinal capillary endothelial cells and pericytes were exposed to low and high glucose concentrations, and immunoblot analysis, 14C-3-O-methylglucose transport activity, and cytochalasin B binding assays were used to characterize the glucose transporters. RESULTS:GLUT1, but not GLUT3 or GLUT4 transporter isoforms, was present in plasma membranes isolated from each cell type. The EC50 for glucose transport was similar in endothelial cells and pericytes (3.94 to 0.48 mM versus 2.24 to 0.69 mM) and was consistent with the EC50 previously reported for GLUT1 transporters on other cells, as was the observation that insulin did not acutely stimulate glucose transport in either cell type. The Vmax for glucose transport was greater in pericytes than endothelial cells (71 to 25 versus 14.5 to 0.8 pmol/10 s/g DNA). Exposure of pericytes to 20 mM glucose for 8 days decreased the initial maximal rate of glucose transport by 30%, compared to pericytes cultured in 5 mM glucose (187 to 7 versus 133 to 9 fmol/20 s/g DNA, P < 0.01), but had no effect on glucose transport activity in endothelial cells. Culture in high glucose decreased the apparent amount of immunoreactive pericyte plasma membrane GLUT1 in immunoblots (0.611 to 0.055 versus 1.0 relative density units), decreased the binding of 3H-cytochalasin B to pericyte plasma membranes, and decreased the mRNA level for GLUT1 in pericytes by 25%. CONCLUSIONS: High-glucose concentrations downregulate glucose transport activity and GLUT 1 content in retinal capillary pericytes but not in endothelial cells. This effect occurred at a pretranslational level. The selective effects of high-glucose concentrations on retinal capillary pericytes in culture might be related to the selective effects of hyperglycemia on these cells in vivo.