Abstract
The major objective of this investigation was to determine the thiol status of chondrocytes and to relate changes in the level of glutathione and cysteine to maturation of the cells as they undergo terminal differentiation. Chondrocytes were isolated from the cephalic portion of chick embryo sterna and treated with all-trans retinoic acid for one week. We found that the addition of 100 nM retinoic acid to the cultures decreased the intracellular levels of glutathione and cysteine from 6.1 to 1.6 and 0.07 to 0.01 nmol/microgram 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 characterize 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 these enzymes and the cytosolic isoenzyme of superoxide dismutase. We probed the specificity of the thiol response by using immature caudal chondrocytes. Unlike the cephalic cells, retinoic acid did not change intracellular glutathione and extracellular cysteine levels, although the retinoid caused a reduction in the intracellular cysteine concentration. Finally, we explored the effect of medium components on chondrocyte thiol status. We noted that while ascorbate alone did not change cell thiol levels, it did cause a 4-fold decrease in the extracellular cysteine concentration. When retinoic acid and ascorbic acid were both present in the medium, there was a marked decrease in the level of glutathione. In contrast, the phosphate concentration of the culture medium served as a powerful modulator of both glutathione and cysteine. Results of the study clearly showed that there is a profound decrease in intracellular levels of both cysteine and glutathione and that thiol levels are responsive to ascorbic acid and the medium phosphate concentration. These findings point to a critical role for thiols in modulating events linked to chondrocyte maturation and cartilage matrix synthesis and mineralization.
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