RETINOIC ACID MODULATION OF GLUTATHIONE AND CYSTEINE METABOLISM IN CHONDROCYTES

The major objective of this investigation was to determine the thiol s tatus of chondrocytes and to relate changes in the level of glutathion e and cysteine to maturation of the cells as they undergo terminal dif ferentiation. Chondrocytes were isolated from the cephalic portion of chick embryo sterna and treated with all-trans retinoic acid for one w eek. We found that the addition of 100 nM retinoic acid to the culture s decreased the intracellular levels of glutathione and cysteine from 6.1 to 1.6 and 0.07 to 0.01 nmol/mu g DNA respectively; retinoic acid also caused a decrease in the extracellular concentration of cysteine. The decrease in chondrocyte thiols was dose and time dependent. To ch aracterize other antioxidant systems of the sternal cell culture, the activities of catalase, glutathione reductase and superoxide dismutase were determined. Activities of all of those enzymes were high in the retinoic acid-treated cells; the conditioned medium also contained the se enzymes and the cytosolic isoenzyme of superoxide dismutase. We pro bed the specificity of the thiol response by using immature caudal cho ndrocytes. Unlike the cephalic cells, retinoic acid did not change int racellular glutathione and extracellular cysteine levels, although the retinoid caused a reduction in the intracellular cysteine concentrati on. Finally, we explored the effect of medium components on chondrocyt e thiol status. We noted that while ascorbate alone did not change cel l thiol levels, it did cause a 4-fold decrease in the extracellular cy steine concentration. When retinoic acid and ascorbic acid were both p resent in the medium, there was a marked decrease in the level of glut athione. In contrast, the phosphate concentration of the culture mediu m served as a powerful modulator of both glutathione and cysteine. Res ults of the study clearly showed that there is a profound decrease in intracellular levels of both cysteine and glutathione and that thiol l evels are responsive to ascorbic acid and the medium phosphate concent ration. These findings point to a critical role for thiols in modulati ng events linked to chondrocyte maturation and cartilage matrix synthe sis and mineralization.