Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells

Abstract

Retinal pigment epithelial (RPE) cells selectively phagocytize rod outer segments (ROS) by a process which may be mediated by specific cell surface receptors. We have studied the kinetics of this process using rat RPE cells grown in tissue culture. By cooling RPE cells to 17 degrees C, the binding and ingestion phases of phagocytosis can be separated. Maximum ROS binding with minimum ingestion occurs at 17 degrees C; above 17 degrees C the rate of ingestion increases markedly. Thus it is possible to measure the kinetics of ROS binding to RPE cells at 17 degrees C and of ROS ingestion at 37 degrees C. At 17 degrees C, ROS binding is saturable, both with respect to time and to ROS concentration. ROS ingestion saturates after 4 hr of incubation at 37 degrees C, after which the cells are refractory to further ROS ingestion for 1-2 hr. During this recovery period, rapid digestion of the internalized ROS takes place. Cycloheximide, when present at a concentration (2 x 10(-5) M) which inhibits protein synthesis by 92%, has no effect on ROS phagocytosis or on the recovery of ROS ingestion at 37 degrees C. This suggests that if receptors mediate the ingestion of ROS by RPE cells, they are not degraded after the ROS are internalized. Dystrophic rat (RCS-p+) RPE cells exhibit normal binding, but very limited ingestion of ROS at 37 degrees C. The rate and amount of ROS binding to these cells at 37 degrees C is comparable with that occurring to normal cells at 17 degrees C. These observations support the hypothesis that there are a limited number of receptors which are specific for ROS binding on the surface of normal and dystrophic rat RPE cells.