Proteoglycan biosynthesis in cultures of corneas and corneal stroma cells from adult rabbits

Abstract

The biosynthesis of proteoglycans has been follwed by [ 3H]glucosamine incorporation into rabbit corneal organ cultures and confluent cell cultures of stromal fibroblasts. Proteoglycans were extracted with guanidinium chloride, purified by chromatography on DEAE-cellulose and characterized by gel chromatography. The cell cultures accumulated polysaccharides linearly over 48 hr. Ninety per cent of the polysaccharide material was secreted into the medium. The main part of this constituted high-molecular weight hyaluronate but a proteoglycan fraction containing mostly dermatan sulphate was also found. The cells contained proteoglycans which mainly contained heparan and dermatan sulphates. The amount of keratan sulphate was insignificant in the cell cultures. The time course of synthesis in the organ cultures showed a discontinuous pattern with a rapid initial phase during the first two hours and after that a slower incorporation. The organ cultures synthesized proteoglycans containing equal amounts of chondroitin sulphate and a fraction which was presumably keratan sulphate (the two dominating polysaccharides of normal cornea). However, even these cultures synthesized mainly heparan sulphate and dermatan sulphate proteoglycans. Less proteoglycan material was secreted into the medium than in the cell cultures. The changes in the biosynthetic pattern when corneas are cultivated in vitro have certain features in common with those occurring in injured corneas.