| Abstract: | Conditions were established for short-term primary suspension culture of chondrocytes from the Swarm rat chondrosarcoma. Proteoglycan and hyaluronate synthesis on Day 0 to Day 2 in culture was investigated and compared with that for plated cultures. Incorporation of 35S]sulfate into proteoglycans was the same for both suspension and plated cultures. 35S-Proteoglycan synthesis decreased by about 80% between Days 0 and 1 irrespective of culture conditions. Suspension culture chondrocytes synthesized proteoglycans which were very similar to those made in plated cultures, with respect to hydrodynamic size, glycosaminoglycan, chain length, and composition. 3H]Hyaluronate synthesis accounted for 18 and 23% of the total 3H-glycosaminoglycans synthesized from 3H]glucosamine by suspension and plated cultures, respectively. Suspension culture chondrocytes responded to exogenous hyaluronate (1 mg/ml) by reducing their 35S-proteoglycan synthesis by about 50%. 3H]Hyaluronate synthesis was inhibited by 13% under these conditions. The inhibition was dependent on the concentration of exogenous hyaluronate and reached a plateau level within 2 h. Plated chondrocyte cultures showed little or no response to hyaluronate. Suspension cultures of chondrocytes were prelabeled with 3H]lysine and lysed, and a heavy membrane fraction (12,000g) was extracted with the detergent 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. A Sepharose-hyaluronate affinity gel was used to show that the extract contained hyaluronate binding 3H-labeled proteins and evidence was obtained suggesting that these came from the external face of the plasma membrane. |
