| Popis: |
The synthesis and secretion of glycosaminoglycans (GAGs) in primary cultures of feline retinal pigment epithelium (RPE) was measured. After 14 days in culture, the cells were incubated for 3 days with media containing 20 microCi/ml 35SO4 and 10 microCi/ml [3H]-glucosamine. The GAGs were precipitated from the media and cell layer with cetylpyridinium chloride and ethanol, separated in 0.1 M Ba acetate by cellulose acetate electrophoresis, visualized by Alcian blue staining and fluorography, and quantitated by scanning densitometry and liquid scintillation counting. The predominant radioactively-labeled GAG secreted into the media by RPE cultures was chondroitin sulfate (CS), which accounted for 63% of the 35SO4, and 54% of the 3H incorporated. Radioactively-labeled heparan sulfate (HS), dermatan sulfate (DS), and hyaluronic acid (HA) were also identified in the media. Heparan sulfate accounted for 24% of the 35SO4 and 22% of the 3H, DS contained 13% of the 35SO4 and 7% of the 3H, and HA accounted for 17% of the 3H incorporated into the media GAGs. In contrast, fibroblast cultures secreted primarily HA, which accounted for 84% of the 3H in the media GAGs. The profile of GAGs retained by the cell layer was different from that of the secreted GAGs. The predominant radioactively-labeled GAG associated with the cell layer was HS. This GAG contained 54% of the 35SO4, and 52% of the 3H incorporated into cell layer GAGs. Dermatan sulfate contained 23% of the 35SO4, and 23% of the 3H, while CS accounted for 23% of the 35SO4, and 18% of the 3H incorporated into cell layer GAGs. The remaining 7% of the incorporated 3H was associated with HA. Total GAG profiles were determined for primary cultures of feline RPE by Alcian blue staining, and compared with those of freshly isolated cell samples. The GAG profiles were similar in cultured and freshly-isolated samples; however, the percentage of HS in the freshly-isolated samples was about twice as high as the percentage of HS in cultured samples, while the proportion of DS in cultured samples was higher than in freshly-isolated samples. This study demonstrates that the profile of radioactively-labeled GAGs secreted by primary cultures of feline RPE is distinct from that retained by the cell layer. In addition, the total GAG profile of cultured RPE is similar, but not identical, to that of freshly-isolated cells. |
