Mannose-sensitive HRP endocytosis by the retinal pigment epithelium

Mannose-sensitive endocytosis by rat retinal pigment epithelium (RPE) explants was characterized using the mannose-rich glycoprotein horseradish peroxidase (HRP). The number of HRP-containing endosomes in the RPE was morphologically quantitated by light microscopy while the amount of HRP ingested was biochemically quantitated by enzyme assay. HRP internalized via a mannose-sensitive receptor was differentiated from fluid-phase uptake in competitive inhibition studies using D-mannose. Morphological results showed that most HRP-containing endosomes formed within the first 15 min of incubation and showed little increase in number during 4 hr of continued incubation with HRP. In contrast, the biochemical assay showed a steady increase in the amount of HRP in RPE endosomes measured over time. The addition of 10 mM D-mannose to the incubation medium was associated with a significant decrease in both the number of HRP-containing endosomes and the amount of HRP ingested by RPE explants. Values indicate that half of the total uptake of HRP is mediated by a mannose-sensitive receptor while the balance is ingested via non-specific fluid-phase endocytosis.