Quantitative freeze-fracture and filipin-binding study of retinal pigment epithelial-cell basal membranes in diabetic rats

Breakdown of the blood-retinal barrier in diabetes may be related to alterations in the retinal pigment epithelial (RPE) cell layer. Morphological studies suggest increased permeability of diabetic RPE plasma membranes, and proliferation and fiattening of the RPE basal infoldings have been observed in diabetic animals. In order to determine whether these phenomena are associated with changes in membrane protein or sterol composition, we have used quantitative electron-microscope freeze-fracture and filipin-binding techniques to study the RPE basal membrane in streptozotocin diabetic and 3-O-methyl glucose control rats. Perfusion-fixed retinas were processed for freeze-fracture and filipin-binding analysis. Filipin, a polyene antibiotic, binds specifically to 3-β-hydroxy-sterols to produce membrane deformations recognizable by freeze-fracture. These analyses revealed an 11% increase in the density of intramembrane particles within the cytoplasmic (P-face) leaflet in diabetic rats as compared with the controls (P < 0·01, t test). The increase occurred primarily in 6-9-nm particles, while smaller particles were decreased (P < 0·001, chi-square test). Filipin binding was the same in both groups. These results suggest that alterations in intrinsic membrane proteins may contribute to permeability and surface area changes in the diabetic RPE but that RPE membrane sterols are not affected by diabetes.