Adenine nucleotide metabolism by chondrocytes in vitro: role of ATP in chondrocyte maturation and matrix mineralization.

Abstract

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse-phase high-performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism.