Lectin-Ferritin Binding on Spontaneously Diabetic and Control Rat Retinal Microvasculature

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Abstract
We previously reported, in the spontaneously diabetic Bio-Breeding (BB) rat, an increase in horseradish peroxidase (HRP) uptake that was associated with reduction and patching of cationized ferritin (CF) binding to anionic sites on the luminal plasma membrane of the retinal capillary endothelium (1). To see whether alterations in the negatively charged terminal sugar residues, N-acetyl-glucosamine (NAG) and sialic acid (SA), might contribute to these changes in the diabetic rat retina, we used lectin-ferritin (Fe) conjugates to study the distribution of these sugars on the retinal endothelial luminal membranes. Wheat germ agglutin (WGA, binds to NAG and SA) and Limax flavius (LFA, binds only SA) were used. Plasma membrane WGA-Fe binding was dense and uniform in control animals. Binding sites were also found in coated luminal vesicles, within some uncoated luminal vesicles and on their diaphragms. Unlabeled uncoated luminal vesicles were also seen, suggesting two populations of uncoated vesicles. In diabetic animals, the binding sites were present within the same membrane associated microdomains as in the controls. However, in the majority of outer plexiform layer (OPL) vessels in diabetic animals, WGA-Fe binding was reduced to a single, discontinuous layer of particles (p<0.02). In both diabetic and control vessels, WGA-Fe binding was greatly reduced by the addition of competitive sugars. A few particles remained on the plasma membrane, on the diaphragms of some vesicles, and at the edge of vesicles. LFA-Fe binding was similar to that seen with WGA-Fe in the presence of competitive sugars. These results suggest that luminal membranes of retinal capillaries are rich in NAG and contain little SA. The sparse WGA-Fe binding pattern in the diabetic OPL may reflect decreases in number or accessibility of NAG residues, since similar binding patterns are seen in both the control and diabetic animals under conditions specific for SA. Thus, alteration of terminal NAG residues may contribute to decreased luminal surface anionic sites and increased pinocytotic transport in the retinal microvasculature of spontaneously diabetic BB rats.