Proteoglycan synthesis in suspension cultures of Swarm rat chondrosarcoma chondrocytes and inhibition by exogenous hyaluronate.
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.
Bansal
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Ward
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Mason
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D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002356:Cartilage / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002813:Chondrosarcoma / Q000378:metabolism; D002846:Chromatography, Affinity; D003470:Culture Media / Q000032:analysis; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats
D002352:Carrier Proteins; D003470:Culture Media; D018960:Hyaluronan Receptors; D009363:Neoplasm Proteins; D011509:Proteoglycans; D006820:Hyaluronic Acid