Glycosaminoglycan synthesis and secretion by bovine retinal capillary pericytes in culture

The synthesis and secretion of glycosaminoglycans (GAGs) was characterized in subcultures of bovine retinal capillary pericytes. The GAGs were metabolically labeled with [3H]glucosamine and 35SO4 for 3 days, and then precipitated from the cell layer or media by cetylpyridinium chloride and ethanol, separated by cellulose acetate electrophoresis and further identified by their susceptibility to degradative procedures. The predominant radioactively labeled GAG associated with the pericyte-cell layer was heparan sulfate (HS). Radioactively labeled chondroitin sulfate (CS) and hyaluronic acid (HA) were also present in the pericyte-cell layer. No radioactively labeled dermatan sulfate (DS) was detected. The profile of radioactively labeled GAGs secreted by pericytes into the media differed considerably from that associated with the cell layer. Equal amounts of radioactivity were incorporated into HS and CS. Small quantities of radioactively labeled HA were also present in the media. Although no radioactively labeled DS was detected in the pericyte-cell layer, it was present in the media. The total pericyte-cell layer GAG profile was determined by scanning densitometry of the three bands resolved after cellulose acetate electrophoresis and Alcian Blue staining. The slowest band was identified as HS, and accounted for 17% of the total GAGs. The middle band was identified as DS, and accounted for 34% of the total GAGs. The fastest band was tentatively identified as either DS or chondroitinase AC-resistant CS, and constituted 49% of the total GAGs. The GAGs associated with the fibroblast-cell layer and secreted into the media by fibroblasts also were characterized and compared with those produced by pericytes. The major differences were in the secretion of large amounts of HA into the media by fibroblasts, and the presence of radioactively labeled DS in the cell layer of fibroblasts.