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

Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high t ransepithelial resistance. Cell cultures were maintained on Millicell-PCF c ulture plates, which allow separation of culture medium exposed to apical a nd basal epithelial surfaces. Following various times in culture, apical an d basal culture media were sampled at three day intervals and the glycosami noglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglyca ns were ethanol precipitated, and subjected to hyaluronidase SD and chondro itinase ABC digestion to release hyaluronan and chondroitin sulfate disacch arides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band qu antitated, Hgaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001). In con trast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin a nd 6-sulfated chondroitin disaccharides in apical and basal media was non-p olar Confocal microscopy of cultures probed with a hyaluponan-specific fluo rotag established that the HA evident in these cultures is restricted to th e apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted prefe rentially from the apical surface, suggesting that this tissue is an import ant source of hyaluronan present in the interphotoreceptor matrix.