Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface

Abstract

Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance. Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces. Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated. Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001). In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar. Confocal microscopy of cultures probed with a hyaluronan-specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix.