Purchase this article with an account.
D Bok, P Senanayake, A Calabro, JG Hu, JG Hollyfield; Glycogen Synthesis in Human RPE . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4569.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the synthesis of glycogen by the retinal pigment epithelium (RPE). Methods: Human RPE was cultured on Millicell- [PCF] culture plates in medium containing 1 mg/ml glucose and the medium was changed every three days. Efficiency of separation of the apical and basal compartments was determined by transepithelial resistance measurements. Cells with their associated matrices (CM), the apical and basal media were collected separately 6 h after the addition of medium on day 49, digested with proteinase K, and ethanol precipitated. Equal aliquots of supernatant and precipitate were treated as follows: 1) 2-aminoacridone (AMAC) derivatized directly to identify those endogenous saccharides with free reducing aldehydes groups, 2) digested with hyaluronidase SD and chondroitinase ABC to identify, characterize and quantitate hyaluronan, chondroitin and dermatan sulfate disaccharides or 3) alkaline phosphatase to confirm the identity of phosphate esters, or 4) digested with glucoamylase to determine total glycogen. The digestion products were fluorotagged with AMAC and separated by electrophoresis. The bands were digitized and their intensities quantified. Results: Glucose decreased by 45% in the apical medium and 36% in the basal medium. Glycogen and glucose derivatives (glucose-6-P, mannose-6-P, glyceraldehyde-3-P, maltose, maltotriose, maltotetraose and unresolved maltooligosaccharides) were present in the CM, maltooligosaccharides were the predominant components. In the apical and basal media mannose was predominant, maltose and phosphate esters were also present, but mannose-6-P was preferentially in the basal medium. In addition, the apical and basal media contained unsulfated and sulfated (4S and 6S) chondroitins and their distribution was similar in apical and basal compartments. Conclusions: This study demonstrates that the glycogen derivatives and the chondroitins synthesized and secreted by the RPE cultures have a distinct profile compared to the saccharides retained in the CM.
This PDF is available to Subscribers Only