Human ARPE-19 cells were maintained at 37°C in a humidified
chamber of 5% CO2 in RPMI 1640 medium containing
5 mM glucose, supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100 μg/mL streptomycin. The culture medium was
replaced with fresh medium every other day. At confluence, cultures
were passaged by dissociation in 0.05% (wt/vol) trypsin in
phosphate-buffered saline (PBS; pH 7.4). After trypsinization, the
cells were seeded at a density of 1.9 × 105 cells/well in 24-well culture plates and cultured in the presence of 2
mL/well of medium. When cultures reached 80% confluence, they were
exposed to either high- or low-glucose medium, after which the uptake
of radiolabeled compounds was measured, as described in a later
section. In most experiments, the high-glucose RPMI 1640 medium
contained 45 mM glucose and the low-glucose medium contained 5 mM
glucose plus 40 mM mannitol. Mannitol was added to control for osmolar
effects. In dose–response experiments, RPMI 1640 medium containing
either 15, 25, 35, or 45 mM glucose was used, and the control medium
contained 5 mM glucose plus 10, 20, 30, or 40 mM mannitol,
respectively. In time-course studies, the exposure to high glucose was
for 4, 6, 9, 12, or 24 hours.